Alu RNA pseudoknot alterations influence SRP9/SRP14 association

Abstract

There are over 1 million Alu elements in the human genome which can be transcribed into discrete, RNA polymerase III transcribed noncoding Alu RNAs. These Alu RNAs often interact with and are regulated by the protein heterodimer SRP9/SRP14. This interaction is dependent on a 5′ pseudoknot domain in the Alu RNA that is thought to be held together by a canonical nucleotide triad within a U-turn motif. Herein, we discover a significant reduction in BC200 expression after mutation of a critical guanosine in the U-turn motif within its pseudoknot domain. We studied a recently discovered short human Alu RNA, EB120, which lacks the canonical Alu RNA U-turn nucleotide triad. We tested the expression of EB120 in 18 different human cell lines and tissues. EB120 was found to lack association with SRP9/SRP14 in a cellular context. Small-angle X-ray scattering followed by atomistic computation structure prediction suggests the BC200 Alu domain and its U-turn mutant both possess a canonical Alu RNA fold, while EB120 lacks one. Our results highlight the structural diversity of Alu RNA, and the impact mutations may have on Alu RNA function.

INTRODUCTION

Alu elements are primate-specific short interspersed elements (SINEs), which make up ∼10% of the human genome (Lander et al. 2001). There are over 1 million copies of human Alu elements, with 99% being unique in sequence (Umylny et al. 2007). Alu elements are ∼300 base pair retrotransposons, propagated through the enzymatic activity of ORF2, a protein with endonuclease and reverse transcriptase domains (Deininger and Daniels 1986; Dewannieux et al. 2003). These elements are believed to be active in humans with estimates ranging from one new Alu insertion in every 20–200 births (Deininger and Batzer 1999; Cordaux et al. 2006; Xing et al. 2009; Feusier et al. 2019). Insertions occur throughout the genome but especially near gene-rich regions, with Alu elements existing in introns, protein-coding exons, and noncoding regions (Häsler et al. 2007; Chen and Yang 2017). Dimeric Alu elements are defined by a left-arm monomer (∼120 nt), an internal polyadenosine tract, a right-arm monomer (∼150 nt), and a terminal polyadenosine tract (Dewannieux et al. 2003; Gussakovsky and McKenna 2021). Alu elements possess internal RNA polymerase III promoters (A- and B-boxes) and can be transcribed as noncoding, class II RNA polymerase III transcripts (Geiduschek and Tocchini-Valentini 1988). Alu elements are also extremely abundant on RNA polymerase II transcribed genes, with an average of ∼16 Alu elements found in each pre-mRNA (DeCerbo and Carmichael 2005).

Alu elements have significantly contributed to primate evolution by increasing genetic diversity through retrotransposition, as well as providing novel sources of alternative splicing (Makałowski et al. 1994; Sorek et al. 2002; Jurka 2004; Cordaux and Batzer 2009; Daniel et al. 2014). Alu elements are not well expressed, with an estimated 99% epigenetically repressed by methylation, or lacking appropriate upstream RNA polymerase III elements required to enhance transcription (Liu et al. 1994; Roy et al. 2000). Initial estimates of Alu RNA abundance suggested 100–1000 cytoplasmic copies of Alu RNA per HeLa cell (Liu et al. 1994). Difficulty in differentiating and uniquely identifying RNA polymerase III transcribed noncoding Alu RNA from other abundant Alu and Alu-like RNA has also raised challenges in understanding the breadth of Alu RNA expressed (Paulson and Schmid 1986; Gussakovsky and McKenna 2021). Unique RNA polymerase III Alu transcripts have since been identified using next-generation sequencing, but are limited to Alu RNA that could be uniquely mapped while also considering the error rates associated with sequencing (Conti et al. 2015).

Alu elements are thought to have been derived from the 299 nt 7SL RNA, the RNA component of the signal recognition particle, which regulates ∼30% of the human proteome (Ullu and Tschudi 1984; Akopian et al. 2013). In humans, the signal recognition particle is composed of six proteins (SRP9/SRP14 heterodimer, SRP19, SRP54, and SRP68/SRP72 heterodimer) and scaffolded by 7SL (Politz et al. 2000). The human 7SL can be segregated into three distinct regions: a 100 nt 5′ end, central 155 nt section, and a 45 nt 3′ end. The 100 nt 5′ end and 45 nt 3′ end together form a sequence homologous to the human right Alu monomer (Ullu et al. 1982). The central 155 nt region of the human 7SL is homologous to nonprimate 7SL, suggesting 7SL is more ancient than Alu elements (Ullu and Tschudi 1984). Alu elements are believed to have been generated by the processing of 7SL into fossil Alu monomers, followed by deletions to form the left arm and right arm monomers (Quentin 1992b). A fusion between a left and right Alu monomer resulted in the formation of the dimeric Alu element (Quentin 1992a). Fossil Alu monomers persist in primate genomes, with some displaying retrotransposition capabilities (Kojima 2011). Dimeric Alu RNA can also be processed into monomeric left arm monomers called small cytoplasmic Alu RNA (scAlu) (Maraia et al. 1993; Chang and Maraia 1993; Chang et al. 1994, 1996; Shaikh et al. 1997).

Alu RNAs are part of a homologous but diverse family, made of three main families from most ancient to youngest AluJ, AluS, and AluY, encompassing 225 different subfamilies (Price et al. 2004). The first ∼50 nt of Alu RNA/7SL are evolutionarily conserved and correspond to the SRP9/SRP14 binding site (Labuda and Striker 1989; Strub et al. 1991; Birse et al. 1997; Weichenrieder et al. 1997, 2000, 2001). This region in Alu RNA is believed to mimic the 7SL 5′ terminus, with the conservation of secondary and tertiary structures required for SRP9/SRP14 binding and the formation of a 5′ pseudoknot domain (Huck et al. 2004). Appropriate 7SL secondary structure is required for efficient transcription of 7SL (Emde et al. 1997). It was recently demonstrated that SRP9/SRP14 is in close proximity to the 7SL genomic locus and positively impacts its transcription (Gussakovsky et al. 2023). SRP9/SRP14 binding is also critical for trafficking, localization, and post-transcriptional processing of 7SL (He et al. 1994; Chen et al. 1998; Grosshans et al. 2001; Perumal et al. 2001; Sommerville et al. 2005). Studies of other Alu RNA have begun to demonstrate the importance of specific nucleotides in the first ∼50 nt of Alu RNA as a prerequisite for SRP9/SRP14 binding and subsequent functions such as alternative splicing (Borovská et al. 2023). SRP9/SRP14 binding to Alu RNA is also functionally important for retrotransposition, scAlu RNA generation, global translation, and the modulation of stress responses (Sarrowa et al. 1997; Dewannieux et al. 2003; Bennett et al. 2008; Gussakovsky et al. 2024).

The best-studied Alu and Alu-like RNAs are the 7SL and BC200 RNAs. BC200 is a 200 nt anthropoid primate-specific RNA and the transcription product of BCYRN1 (Watson and Sutcliffe 1987; Martignetti and Brosius 1993). BC200 is characterized by a sequence homologous to a free left arm Alu monomer-Cl group (FLAM-Cl) in its first 120 nt, followed by a 40 nt adenosine-rich and a 40 nt variable region with a 12-cytosine tract (Martignetti and Brosius 1993; Tiedge et al. 1993). Two of the validated protein binding partners of BC200 are SRP9 and SRP14 (Booy et al. 2018). Binding studies have previously shown SRP9/SRP14 binds BC200 at an affinity similar to 7SL (Bovia et al. 1997; Kremerskothen et al. 1998). As observed for 7SL, SRP9/SRP14 is in close proximity to the BC200 genomic locus and positively regulates its transcription (Gussakovsky et al. 2023). BC200 is expressed in high levels in brain tissue, but is aberrantly expressed in multiple tumor cell types (Chen et al. 1997; Iacoangeli et al. 2004; Hu and Lu 2015; Booy et al. 2017; Samson et al. 2018). Poor cancer prognosis is correlated with BC200 expression, and the RNA is critical for tumor cell viability and migration (Iacoangeli et al. 2004; Zhao et al. 2016; Booy et al. 2017). BC200 has previously been presented as both a negative and positive translational regulator, dependent on biological context (Kondrashov et al. 2005; Lin et al. 2008; Jang et al. 2017; Booy et al. 2021). Recently, BC200 has been determined to be stably processed into its left arm Alu monomer (BC120) (Booy et al. 2024). The varying ratio between BC200 and BC120 in different cell lines may explain the previous conflicting reports on BC200-mediated translation regulation (Booy et al. 2024).

The canonical Alu RNA fold has been described extensively from the human 7SL in complex with SRP9/SRP14 or as part of the signal recognition particle as a τ-junction, or two stem–loops separated out by a U-turn motif (Birse et al. 1997; Bovia et al. 1997; Weichenrieder et al. 2000, 2001; Jomaa et al. 2022). Additional structural elucidations of 7SL include high-resolution structures of the signal recognition particle RNA Alu domain originating from bacterial, archaeal, or yeast organisms (Strub et al. 1999; Bousset et al. 2014; Kempf et al. 2014; Soni et al. 2021). On the other hand, investigations into the structural diversity of human Alu RNA have been limited. Other human Alu RNAs previously characterized include low-resolution solution structures of the sense and antisense Alu inverted repeats in the human LincRNA-p21, and a high-resolution structure of the consensus human AluJ RNA in the context of retrotransposition (Ahl et al. 2015; D'Souza et al. 2022). In both cases, there is a description of a canonical Alu RNA fold, which requires a specific base stack formed by a critical nucleotide triad in the U-turn motif (Weichenrieder et al. 2000; Huck et al. 2004; Ahl et al. 2015). Mutation of a U-turn guanosine in 7SL and other Alu RNA leads to attenuation of SRP9/SRP14 association and significantly impacts different biological contexts such as retrotransposition and translation (He et al. 1994; Chang et al. 1997; Bennett et al. 2008; Ivanova et al. 2015; Gussakovsky et al. 2024). Furthermore, studies into the structure of BC200 have been limited to studies such as RNase digestions, or bioinformatic analyses (Jung et al. 2014; Booy et al. 2016; Sosinska-Zawierucha et al. 2018). Herein, we study the functional impact of the mutation of a BC200 Alu domain U-turn motif guanosine. A newly discovered human Alu RNA, EB120, that lacks the canonical U-turn nucleotide triad was also studied and found to not associate with SRP9/SRP14. This was followed up with the biophysical and low-resolution structural characterization of the BC200 Alu domain, and EB120. Our study demonstrates the discovery of a novel RNA polymerase III transcribed noncoding human Alu RNA, and differing examples of Alu RNA folds.

RESULTS

BC200 and 7SL U-turn motif mutants associate with SRP9/SRP14 in lysate

As conservation of the Alu RNA U-turn motif is critical for downstream SRP9/SRP14 biological processes (He et al. 1994; Chang et al. 1997; Bennett et al. 2008; Ivanova et al. 2015; Gussakovsky et al. 2024), we sought to corroborate these findings with the BC200 Alu domain. Our previous work demonstrated that the BC200 Alu domain structure is preserved with the removal of the terminal 3′ region (resulting in BC120) (Booy et al. 2016). BC120 is also stable and functional in a cellular context (Booy et al. 2024). To determine whether conservation of the BC120 U-turn nucleotide triad (G5, U24, G25) was important for the interaction with SRP9/SRP14 (Fig. 1A), we purified in vitro transcribed BC120, BC120 G25C, 7SL, and 7SL G24C. During the quality control assessment of the RNAs, we subjected the purified products to denaturing urea Tris-borate EDTA (TBE) gels and found the corresponding RNAs migrating similarly (Fig. 1B,C, left-most gel). When separating the RNAs on a native TBE gel, BC120 G25C and 7SL G24C migrated modestly slower than their wild-type counterparts (Fig. 1B,C, right-most gel). Furthermore, 7SL and 7SL G24C had multiple conformations observed of similar size. The RNAs were then biotinylated and incubated in a HEK293T cytoplasmic lysate prior to a streptavidin pulldown. A control streptavidin pulldown was performed with no addition of RNA and was used to assess the specificity of the beads. These samples were then analyzed by western blot with antibodies specific to SRP9, SRP14, and ACTB (Fig. 1D). Surprisingly, SRP9 and SRP14 proteins were significantly enriched by both wild-type and U-turn motif mutants. Higher levels were observed from BC120/BC120 G25C compared to 7SL/7SL G24C, and increased enrichment from 7SL G24C relative to 7SL. The beads only sample did not enrich for SRP9 or SRP14 indicating that the pulldowns were specific. ACTB was a negative control and was not pulled down by either RNA, indicating specific interactions. Biotinylation efficiency for each RNA was similar, as demonstrated by a slot blot of each RNA with detection by streptavidin-HRP (Fig. 1D, bottom panel). These results indicate that using streptavidin pulldowns, U-turn mutants have similar levels of association with SRP9/SRP14 in lysate.

FIGURE 1.

Conservation of the BC200 U-turn nucleotide triad is critical for expression. (A) Predicted secondary structure of BC200 Alu domain as previously demonstrated by BC120 (Booy et al. 2024). Highlighted in red is G25, which corresponds to the U-turn guanosine in 7SL when mutated impacts SRP9/SRP14 binding (Chang et al. 1997). The G25C label refers to the mutation of this guanosine to a cytosine. (B) Quality assessment of in vitro transcribed RNA. Left panel is a denaturing Tris-borate EDTA (TBE) 12% polyacrylamide gel electrophoresis of 1 µg BC120 and BC120 G25C. Gel was stained with toluidine blue. A single dominant band is present for both RNAs indicating a single RNA species. Right panel is the same as the left panel but for native TBE 12% polyacrylamide gel electrophoresis. (C) Same as (B) but for 7SL and 7SL G24C on denaturing TBE 6% polyacrylamide gel electrophoresis (left panel) and native TBE 6% polyacrylamide gel electrophoresis. (D) Streptavidin pulldown assay with 3′ biotinylated BC120, BC120 G25C, 7SL, 7SL G24C, or only beads in HEK293T cytoplasmic lysates. Formed RNA–protein complexes were collected with streptavidin magnetic beads, separated by SDS-PAGE, and analyzed by western blot with antibodies specific to SRP9, SRP14, and ACTB. Biotinylation efficiency was demonstrated by a slot blot of 10 pmol of each RNA and detected with streptavidin-HRP. (E) Whole-cell lysate RNA extracted from HEK293T cells transfected with a pcDNA3.1 Hygro(+) vector lacking or containing a BC200 or BC200 G25C expression cassette. Nucleic acids collected were treated with DNase I to digest carryover plasmid prior to repurification. RNA was analyzed by RT-qPCR with 25 ng of RNA per reaction. Primers were specific to BC200. Data represent the mean of three biological replicates measured in duplicate, with error presented as ±standard deviation. P-values were calculated using an unpaired two-tailed t-test with statistical significance indicated by (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. (F) Same as (E) but for hygromycin phosphotransferase (HPH) mRNA.

Conservation of the BC200 U-turn nucleotide triad is critical for expression

The lack of difference in SRP9/SRP14 association after the introduction of a U-turn motif mutation led us to investigate the mutation in a downstream biological process. Since SRP9/SRP14 plays a role in regulating BC200 transcription (Gussakovsky et al. 2023), we sought to understand whether conservation of the U-turn nucleotide triad was necessary for transcription of BC200. We transfected HEK293T cells with a pcDNA3.1 Hygro(+) vector containing a BC200, or BC200 G25C expression cassette. This vector expresses hygromycin phosphotransferase (HPH) to enable resistance toward hygromycin. RNA was assessed by RT-qPCR with primers specific to BC200, and HPH mRNA as a measure of transfection efficiency. Relative to the empty vector, BC200 overexpression led to an 83-fold increase in BC200, while BC200 G25C overexpression only resulted in a 37-fold increase (Fig. 1E). Transfection efficiency of the vectors was consistent as demonstrated by the stable hygromycin phosphotransferase (HPH) levels (Fig. 1F). In summary, the mutation of a U-turn motif guanosine significantly decreased BC200 expression levels.

Discovery of a novel RNA polymerase III transcribed noncoding RNA mapping to an Alu element

To better understand the significance of the canonical Alu RNA U-turn motif nucleotide triad, we sought to identify additional RNA polymerase III transcribed noncoding Alu RNA. Initial screening of BC120 clones detected several additional Alu RNA with roughly 120 nt in length, one of which was detected multiple times across both MCF-7 and brain tissue RNA samples (Booy et al. 2024). This RNA mapped to the left arm monomer of an AluJo element positioned at Chr1: 116709982–116710098 (GRCh38/hg38) according to RepeatMasker and the UCSC Human Genome Browser (Kent et al. 2002; Bao et al. 2015). Using MCF-7 and brain tissue RNA, 12 clones were generated with 5′ RACE. This confirmed the 5′ end of the RNA mapped back to the 5′ end of the AluJo element at Chr1: 116709982 (Supplemental Fig. S1A). Nineteen clones were generated by 3′ RACE where all 19 clones contained the entire AluJo element. Seven clones terminated with three uridines (ending mapped to Chr1: 116710101), five clones terminated with four uridines, and the remaining clones terminated with various uridine tracts or termination at a downstream RNA polymerase III termination signal (Supplemental Fig. S1B). The majority of transcripts terminated with three or four uridines, which is consistent with the canonical RNA polymerase III termination sequence of four thymines. Due to this, we classified the RNA to be 120 nt long, with the first 117 nt consisting of the predicted left arm monomer of an AluJo element, followed by three terminal uridines. In all 5′ and 3′ RACE clones, there were no instances of this sequence embedded in a larger transcript. Since the RNA was identified in brain tissue, we refer to the RNA as Expressed in Brain 120 (EB120) (GenBank accession number PP505732).

As EB120 mapped back to an archetypal AluJo element, we sought to confirm if EB120 was representative of a noncoding Alu gene. BCYRN1 was used for comparison since BCYRN1 is an Alu RNA polymerase III gene that transcribes BC200 (Martignetti and Brosius 1993; Tiedge et al. 1993). For class II RNA polymerase III genes like BCYRN1 and Alu elements in general, promoters are represented as A-box (consensus: RGYNNAGTGG), and B-boxes (consensus: GGTTCRANNCC), which are internal to the transcribed sequence (Englert et al. 2004; Kim et al. 2017). Consistent with other human RNA polymerase III transcripts, the termination sequence is canonically four thymidines (Hamada et al. 2000). For BCYRN1, its A-box is present from +6 to +15 (GGCGCGGTGG), B-box is present from +78 to +88 (AGTTCGAGACC), and the termination sequence is present from +197 to +201 (TTTT) (Fig. 2A). Comparing EB120 to BCYRN1, we observe the same characteristics. The EB120 A-box is present from +5 to +15 (GGACAAGGTGG), B-box is present from +75 to +85 (AGTTCGAGACC), and the termination sequence is present from +117 to +121 (TTTT) (Fig. 2A). To be noted, the B-boxes in BCYRN1 and EB120 are in different nucleotide positions but are identical in sequence. These data suggest that EB120 is an RNA polymerase III transcribed noncoding Alu gene.

FIGURE 2.

EB120 maps to an RNA polymerase III transcribed noncoding Alu gene. (A) Annotated genomic loci of BCYRN1 and EB120. BCYRN1 transcribes BC200 (GenBank accession number NR_001568), and EB120 transcribes EB120 (GenBank accession number PP505732). +1 of BCYRN1 is located at Chr2: 47335315 (GRCh38/hg38), while +1 of EB120 is located at Chr1: 116709982 (GRCh38/hg38). The annotation of the A Box, B Box, and termination sequence is determined from previous studies based on consensus sequences (Hamada et al. 2000; Englert et al. 2004; Kim et al. 2017). The annotations of the FLAM-Cl (+1 to +120) of BCYRN1 and the AluJo (+1 to +117) of EB120 were determined from previous studies, and as according to RepeatMasker and the UCSC Human Genome Browser RepeatMasker (Martignetti and Brosius 1993; Kent et al. 2002; Bao et al. 2015). (B) Frequency distribution of the length of 43,903 human AluJo elements (Bao et al. 2015). The vast majority of AluJo elements were concentrated from lengths of 110–140 nt, which represented monomeric Alu elements, and 270–320 nt, which represented dimeric Alu elements. The red dashed line at 117 nt highlights the group of 786 human AluJo elements with the same length of Alu domains as EB120. (C) Frequency distribution of the sequence identity of 117 nt long AluJo elements relative to the consensus AluJo sequence (Price et al. 2004). The black dashed line represents the median, while the dotted black lines represent 1 SD from the median. The red solid line represents the sequence identity of the AluJo element within EB120 relative to the AluJo consensus. (D) Frequency distribution of the sequence identity of the first 45 nt of 117 nt long human AluJo elements relative to the consensus AluJo element. The red dashed line represents both the median and the sequence identity of the first 45 nt of the AluJo element within EB120 relative to the AluJo consensus. The black dotted lines represent 1 SD from the median. (E) Sequence alignment of the first 117 nt of the human AluJo RNA consensus, AluJo domain of EB120, and FLAM-Cl domain (monomeric precursor of dimeric AluJ elements) of BC200 (Price et al. 2004). The numbering refers to position 1 of the AluJo RNA consensus and the relative alignment of the AluJo domain of EB120 or FLAM-Cl domain of BC200 to this position. Gaps are introduced to maximize the identities. Matches are indicated by dots, while differences are indicated by the specified nucleotide. (F) Predicted secondary structure of EB120 by mapping the primary sequence onto the canonical Alu RNA secondary structure (Labuda and Zitkiewicz 1994). Nucleotides highlighted in red indicate sequence differences compared to the BC200 Alu domain.

To verify whether the sequence of the Alu domain of EB120 was consistent with other AluJo sequences, we analyzed the length and sequence similarity of all 43,903 human AluJo elements obtained from RepeatMasker (Bao et al. 2015). By calculating the length of each AluJo element, we found a total of 786 AluJo elements with a length of 117 nt, which was the second most abundant length of AluJo elements (Fig. 2B). Furthermore, we aligned all 117 nt-long AluJo elements and calculated their sequence identity relative to the first 117 nt of the consensus AluJo (Supplemental Table S1; Price et al. 2004). Compared to the AluJo consensus, AluJo elements have an 84 ± 5% (median ± standard deviation) sequence identity, whereas the EB120 AluJo element has a 79% sequence identity (Fig. 2C). The assessment was repeated with a restriction to the first 45 nt of the Alu element since this is an evolutionarily conserved region (Labuda and Striker 1989; Strub et al. 1991). Compared to the AluJo consensus over the first 45 nt, AluJo elements have an 84 ± 8% sequence identity, while the EB120 AluJo element has an 84% sequence identity (Fig. 2D). The alignment of the AluJo consensus, EB120, and BC120 is presented in Figure 2E. BC120 has an 85% sequence identity over the full-length, and a 93% sequence identity over the first 45 nt to the AluJo consensus, demonstrating the similarities of different Alu elements. We mapped the primary sequence of EB120 onto the canonical Alu RNA secondary structure to generate a predicted secondary structure of EB120 (Fig. 2F; Labuda and Zitkiewicz 1994). This structure predicts EB120 to not have the nucleotide triad present in the canonical positioning in addition to significant differences in base-pairing throughout the structure. Together, these sequence comparisons suggest that the AluJo element in EB120 is a representative AluJo element lacking the canonical U-turn motif nucleotide triad.

EB120 is expressed in specific cell lines and localized in the cytoplasm

Since EB120 was first identified in MCF-7 cells and brain tissue, we sought to determine its breadth and abundance. To achieve this, we performed direct quantification of EB120 by real-time quantitative PCR (RT-qPCR). Due to the high level of similarity between Alu sequences, we used a TaqMan assay with an internal probe in order to increase its specificity. To confirm the TaqMan primer/probe set was specific to EB120, we separated qPCR products out by agarose gel electrophoresis, excised, blunt end cloned, and sequenced to confirm the amplicons were the correct amplification product of EB120 (Supplemental Fig. S2). A linear standard curve was generated by a serial dilution of in vitro transcribed EB120 that spanned at least five orders of magnitude (Fig. 3A). EB120 expression was quantified in RNA extracted from a panel of 10 different immortalized cell lines (MCF-7, SK-BR-3, T-47D, MDA-MB-231, SK-OV-3, A2780, A549, A375, HeLa, HEK293T) and eight different human tissues (brain, ovary, breast, testis, lung, liver, bone marrow, small intestine). Interestingly, only 3/10 cell lines (MCF-7, SK-BR-3, T-47D) and 2/8 tissues (brain, ovary) expressed EB120 within the linear dynamic range (Fig. 3B). The abundance of EB120 (18–350 copies per ng RNA) in the tested samples is significantly lower compared to the range of BC200 (360–1,500,000 copies per ng RNA), and BC120 (1100–750,000 copies per ng RNA) (Booy et al. 2017, 2024). Since EB120 was most abundant in SK-BR-3 cells (Fig. 3B), we investigated EB120 expression levels in SK-BR-3 cells over time. We found that EB120 was highest at 24 h after seeding, with a significant decrease (∼50%) in expression after 48 and 72 h (Supplemental Fig. S3). From these data, it was evident that EB120 was expressed in specific cell lines, but at a lower abundance than BC200 and BC120.

FIGURE 3.

EB120 is expressed in specific cell lines and localized in the cytoplasm. (A) Standard curve generated by RT-qPCR of in vitro transcribed EB120 with murine carrier RNA using a TaqMan assay with a primer/probe set specific to EB120. Each data point represents the mean of three technical replicates, with linear regression performed with GraphPad Prism 9 software to obtain a line of best fit. (B) EB120 expression was assessed in a panel of 10 immortalized cell lines and eight human tissues, with the presented samples expressing within the linear dynamic range. Twenty-five nanograms of total RNA from immortalized cell lines and 50 ng of total RNA from human tissues were used as template for each reaction. Data represented the mean of three technical replicates, with error shown as ±standard deviation. (C) Relative EB120 expression in the indicated subcellular fractions was assessed by RT-qPCR. Twenty-five nanograms of total RNA were used as template from each fraction and relative RNA abundance corrected according to the portion of the total RNA present in each fraction. Data were representative of three biological replicates, presented as the mean of three technical replicates, with error shown as ±standard deviation. (D) As in (C) with primers specific to GAPDH mRNA. (E) As in (C) with primers specific to GRP94 mRNA. (F) As in (C) with primers specific to MALAT1 RNA.

Given that previous studies have identified Alu RNA to be prominently localized in the cytoplasm, we sought to determine the cellular localization of EB120 (Maraia et al. 1993; Liu et al. 1994; Booy et al. 2017, 2024). We performed subcellular fractionation followed by RT-qPCR. RNA was purified and extracted from soluble cytoplasmic, membrane-bound/organelle, nuclear soluble, and chromatin-bound/cytoskeletal fractions from SK-BR-3 cells. EB120 was found to be primarily in the cytoplasmic and membrane-bound/organelle fractions (Fig. 3C). GAPDH mRNA was used as a marker for the soluble cytoplasmic fraction (Fig. 3D; Jagannathan et al. 2011). GRP94 mRNA was used as a marker for the membrane-bound/organelle fractions (Fig. 3E; Pyhtila et al. 2008). MALAT1 RNA was used as a nuclear marker (Fig. 3F; Hutchinson et al. 2007). This experiment suggests that like other Alu RNA, a large proportion of EB120 is localized in the cytoplasm.

SRP9/SRP14 pulldowns do not enrich EB120 relative to BC200 or BC120

The discovery of EB120, an expressed novel RNA polymerase III transcribed noncoding Alu RNA lacking the canonical U-turn nucleotide triad, led us to investigate SRP9/SRP14 association in a cellular context. We performed native RNA coimmunoprecipitations in using antibodies specific to SRP9, SRP14, and an isotype control. Co-immunoprecipitated RNA was analyzed by RT-qPCR with primers specific to BC200, and EB120 (Fig. 4A). While BC200 was enriched with SRP9 (39%) and SRP14 (45%) antibodies, EB120 had almost no enrichment compared to BC200 with either antibody used (SRP9—1%; SRP14—1%). The isotype control poorly enriched either RNA, indicative of specific pulldowns. To confirm EB120 was not transiently interacting with SRP9/SRP14, we repeated the experiment with formaldehyde cross-linking prior to cell lysis. This did not result in any significant increase in the enrichment of EB120 by SRP9 (0.1%), or SRP14 (0.2%) compared to BC200 RNA (SRP9—23%; SRP14—36%) (Fig. 4B). To ensure these results were not due to the low abundance of EB120 relative to BC200, we repeated the native RNA coimmunoprecipitations 24 h after transfection of an empty vector, or a vector containing an EB120 expression cassette. Overexpression led to an ∼50-fold increase in EB120 expression and an increase in enrichment compared to the empty vector for SRP9 (Empty—1%; Overexpression—2%) and SRP14 (Empty—1%; Overexpression—5%) immunoprecipitations. This increase was not comparable to the enrichment of BC200 by SRP9 (Empty—55%; Overexpression—66%), or SRP14 (Empty—70%; Overexpression—82%) (Fig. 4C). As a second method to verify this result, we biotinylated in vitro transcribed BC120 and EB120 and incubated the labeled RNA in a cytoplasmic lysate prior to a streptavidin pull-down and western blot analysis with antibodies specific to SRP9, SRP14, and GAPDH (Fig. 4D). SRP9 and SRP14 were significantly enriched by BC120. In contrast, SRP9 and SRP14 had significantly less enrichment by EB120. GAPDH as a negative control was not pulled down by either RNA, indicating specific interactions. To confirm biotinylation efficiency for each RNA was similar, a slot blot of each RNA with detection by streptavidin-HRP was performed. In summary, using four different pulldown approaches, SRP9/SRP14 did not significantly enrich EB120 compared to BC200 or BC120.

FIGURE 4.

SRP9/SRP14 pulldowns do not enrich EB120 compared to BC200 or BC120. (A) RT-qPCR analysis of native RNA immunoprecipitation of SRP9, SRP14, and an isotype control with a SK-BR-3 cytoplasmic lysate. Primers/probe sets used were specific to BC200 or EB120. Data were representative of three independent biological replicates, represented as the mean of three technical replicates and error shown as ±standard deviation. (B) Same as (A), but with formaldehyde cross-linking prior to lysis. (C) Same as (A), but with transfection of an empty vector, or vector with an EB120 expression cassette. (D) Streptavidin pulldown assay with 3′ biotinylated BC120 or EB120 in SK-BR-3 cytoplasmic lysates. Formed RNA–protein complexes were collected with streptavidin magnetic beads, separated by SDS-PAGE, and analyzed by western blot with antibodies specific to SRP9, SRP14, and GAPDH. Biotinylation efficiency was demonstrated by a slot blot of 10 pmol of each RNA and detected with streptavidin-HRP.

Biophysical characteristics of the U-turn mutant and EB120 are similar to the BC200 Alu domain

Since expression of the BC200 Alu domain U-turn mutant and EB120 association to SRP9/SRP14 were significantly different compared to the BC200 Alu domain, we investigated their biophysical characteristics. In vitro transcribed BC120, BC120 G25C, and EB120 were subjected to thermal denaturation while monitoring absorbance λ at 260 nm. As observed in Figure 5A, the BC120 and EB120 have similar melting profiles and melting temperatures (BC120 T_m = 64°C; EB120 T_m = 63°C). On the other hand, BC120 G25C has a higher melting temperature (T_m = 68°C). In all three cases, the melting profiles are indicative of extensive base-pairing. This study also quantified the hyperchromic shift in the RNA, with both RNA increasing absorbance λ at 260 nm by 24% for BC120, 23% for BC120 G25C, and 24% for EB120 at full denaturation compared to the folded RNA (Puglisi and Tinoco 1989). We also conducted spectropolarimetry on BC120, BC120 G25C, and EB120 to qualitatively assess the secondary structures of the RNAs. From the data collected, BC120, BC120 G25C, and EB120 display similar profiles with a dominant positive peak at 266 nm and a negative peak at 210 nm, consistent with A-form duplex RNA (Fig. 5B; Chauca-Diaz et al. 2015; Booy et al. 2016).

FIGURE 5.

BC120, BC120 G25C, and EB120 have similar biophysical properties. (A) Thermal denaturation of 1 µM BC120, BC120 G25C, and EB120 in PBS, pH 7.4. Absorbance was monitored at 260 nm with temperatures increasing at a rate of 2°C/min and absorbance measured every 10 sec. Data were fit to a sigmoidal curve and its first derivative presented. (B) Circular dichroism spectra of 5 µM BC120, BC120 G25C, and EB120 in PBS, pH 7.4. Spectra were collected at room temperature at a rate of 100 nm/min and averaged over three measurements with a buffer subtraction. (C) Linear extrapolation to infinite dilution of the converted (to standard conditions, pure water at 20°C) apparent sedimentation coefficients s_20°C,w of BC120, BC120 G25C, and EB120 in PBS, pH 7.4. Data were obtained from sedimentation velocity data with direct fitting of s and D to the combined Lamm and Svedberg equations for each sample concentration (Schuck 1998, 2003). Linear regression was performed with GraphPad Prism 9 software to obtain a line of best fit. Error bars represent the 68% confidence interval of each data point, while confidence bands demonstrate the likely location of the true curve with a 90% asymmetrical (profile-likelihood) confidence interval. (D) Linear extrapolation to infinite dilution of the apparent hydrodynamic radii determined by dynamic light scattering of BC120, BC120 G25C, and EB120 in PBS, pH 7.4. Linear regression was performed with GraphPad Prism 9 software to obtain a line of best fit. Each data point represents the mean of 10 technical replicates, with error shown as ±standard deviation. Confidence bands demonstrate the likely location of the true curve with a 90% asymmetrical (profile-likelihood) confidence interval.

To assess the size and shape of the RNAs, we performed sedimentation velocity analyses on BC120, BC120 G25C, and EB120. The c(s, f_r) distributions calculated from the sedimentation profiles suggest that all three RNAs have two distinct species populations within the concentrations measured (Supplemental Figs. S4–S6). We had not previously observed multiple species with size exclusion chromatography and consequently attributed the second species to be a dimer formed by nonspecific interactions (Supplemental Fig. S7). Due to this, we focused the analysis on the monomeric form, while ensuring fitting parameters considered the presence of both species. Direct fitting of the apparent sedimentation coefficient s, and the apparent diffusion coefficient D at each sample concentration to the combined Lamm and Svedberg equations was performed (Fig. 5C; Schuck 1998, 2003). These values were linearly extrapolated to an infinite dilution, and conversion to standard conditions yielded a s°_20°C,w of 4.58 (90% CI 4.30–4.87) S for BC120, 4.46 (90% CI 4.20–4.72) S for BC120 G25C, and 4.30 (90% CI 3.63–4.98) S for EB120. The fitted D values for the monomeric species, and s and D values for the dimeric species for BC120, BC120 G25C, and EB120 samples are shown in Supplemental Figures S8 and S9. To corroborate the notion that BC120, BC120 G25C, and EB120 have similar global sizes, we performed dynamic light scattering experiments on the three RNAs at multiple concentrations. The hydrodynamic radii observed were plotted at each concentration and linearly extrapolated to an infinite dilution to yield an R°_H of 3.41 (90% CI 3.28–3.54) nm for BC120, 3.52 (90% CI 3.39–3.65) nm for BC120 G25C, and 3.16 (90% CI 2.95–3.37) nm for EB120 (Fig. 5D). The data suggest that the biophysical properties of the BC200 Alu domain U-turn mutant and EB120 were not significantly different than the BC200 Alu domain.

BC200 U-turn mutation does not impact Alu conformation

To further characterize the global conformations of the three RNAs, we performed size-exclusion chromatography coupled small-angle X-ray scattering (SEC-SAXS) analyses on BC120, BC120 G25C, and EB120 (Fig. 6A). Guinier analyses were performed on the data sets and determined the radius of gyration (R_G) of BC120 (4.031 ± 0.006 nm), BC120 G25C (4.086 ± 0.009 nm), and EB120 (4.233 ± 0.014 nm) (Fig. 6B; Supplemental Table S2). The linearity of these samples demonstrates that all three RNAs are monodisperse and free of self-association after passing the size-exclusion column (Rambo and Tainer 2011; Burke and Butcher 2012). To investigate whether the RNAs are folded and to determine their conformation, we transformed the intensity data into dimensionless Kratky analysis data (Fig. 6C; Rambo and Tainer 2011). The Kratky analysis indicates that BC120, BC120 G25C, and EB120 are folded molecules with extended conformations based on the relative plateaus of the data sets and non-Gaussian shapes of the plots (Burke and Butcher 2012). The data sets were evaluated using a paired-distance distribution [P(r)] analysis using an indirect Fourier transformation to convert reciprocal space data into real-space electron density data (Burke and Butcher 2012). The P(r) functions revealed the real-space R_G and D_max values for BC120 (4.265 ± 0.005 nm; 13.40 nm), BC120 G25C (4.300 ± 0.007 nm; 13.83 nm), and EB120 (4.299 ± 0.008 nm; 13.60 nm) (Fig. 6D). Real-space R_G values agree with the reciprocal-space values, suggesting the data sets are valid. The shape of the P(r) distributions for both BC120 and BC120 G25C indicates elongated structures, where a Gaussian distribution with maxima present at D_max/2 would be representative of a spherical molecule. BC120 and BC120 G25C have similar maximal dimensions (D_max), while the P(r) distributions are almost identical with one peak with an elongated shoulder at larger radii. Although EB120 has similar D_max to BC120 and BC120 G25C, the P(r) distribution of EB120 has two significant peaks, indicating a distinctive structural feature. This suggests that there is no significant difference in global conformations between BC120 and BC120 G25C, while EB120 has a substantial difference in global conformation.

FIGURE 6.

Small-angle X-ray scattering characterization of BC120, BC120 G25C, and EB120. (A) Scattering data of BC120, BC120 G25C, and EB120 in PBS, pH 7.4. (B) Guinier plots of BC120, BC120 G25C, and EB120 allow the determination of R_G from the low-angle region data and to demonstrate the homogeneity of the samples. (C) Dimensionless Kratky plots of BC120, BC120 G25C, and EB120 depicting extended structures as determined by the non-Gaussian, leveled plateau shape of the curves. (D) Normalized pair distance distribution plots for BC120, BC120 G25C, and EB120, which allows for determination of real-space R_G derived from the scattering data set and includes each RNA's D_max. The curves also highlight near identical shapes for BC120 and BC120 G25C but differs significantly for EB120.

We generated 30 SEC-SAXS envelopes for BC120, BC120 G25C, and EB120 using the DAMMIN software, with favorable agreement of the models represented by χ² values of 1.12 (BC120), 1.12 (BC120 G25C), and 1.11 (EB120). We calculated the normalized spatial discrepancy (NSD) to confirm the agreement of the low-resolution models with the averaged representative (BC120: 0.63 ± 0.01; BC120 G25C: 0.67 ± 0.01; EB120: 0.84 ± 0.03). We also validated the models by using the program HYDROPRO and finding agreement with the predicted model and experimentally determined hydrodynamic parameters (Table 1). The averaged, single-representative SEC-SAXS envelopes of BC120 and BC120 G25C were all extended shapes rather than globular (Fig. 7A,B). Consistent with the P(r) distribution, the SEC-SAXS envelopes of BC120 and BC120 G25C are very similar. The SEC-SAXS envelopes of BC120/BC120 G25C are asymmetrical, with a prominent protrusion located on one end, and a bulge on the other end. From the 90° rotation, it is evident that BC120 and BC120 G25C possess similar D_max. The averaged, single-representative SEC-SAXS envelope of EB120 has an extended shape and rotational symmetry in the y-axis, with two prominent protrusions located on its ends (Fig. 7C), a significant difference compared to BC120 and BC120 G25C. Our data indicate that BC120 and BC120 G25C have similar solution conformations that are significantly different to that of EB120.

TABLE 1.

Experimental and predicted hydrodynamic parameters of BC120, BC120 G25C, and EB120

	BC120		BC120 G25C		EB120
Parameter	Experimental	Model-basedd	Experimental	Model-basedd	Experimental	Model-basedd
s°20°C,w (S)a	4.58 (4.30–4.87)	3.97 [3.85–4.11]	4.46 (4.20–4.72)	4.09 [4.00–4.17]	4.30 (3.63–4.98)	4.02 [3.95–4.05]
RH (nm)b	3.41 (3.28–3.54)	3.69 [3.56–3.70]	3.52 (3.39–3.65)	3.58 [3.51–3.66]	3.16 (2.95–3.37)	3.64 [3.61–3.70]
RG (nm)c	4.265 ± 0.005	4.24 [3.88–4.42]	4.300 ± 0.007	4.06 [3.85–4.27]	4.341 ± 0.008	3.86 [3.76–4.10]
Dmax (nm)c	13.40	13.62 [12.72–14.02]	13.83	14.30 [14.00–14.57]	13.60	12.89 [12.37–13.59]
χ2	–	1.12	–	1.11	–	1.11
NSD	–	0.63–0.01	–	0.84 ± 0.03	–	0.84±0.03

Parentheses indicate the error as a 90% confidence interval. Brackets indicate the range of values. Error represented as a single value indicate ± standard deviation.

^aExperimentally determined from sedimentation velocity data, with error obtained from linear regression analysis.

^bExperimentally determined from DLS data, with error obtained from linear regression analysis.

^cExperimentally determined from P(r) analysis by GNOM (Svergun 1992).

^dModel-based parameters calculated from HYDROPRO, with values representing the average from five models (Ortega et al. 2011).

FIGURE 7.

BC200 Alu domain has a canonical Alu fold while EB120 lacks one. (A) Low resolution SEC-SAXS conformation and structure prediction modeling of BC120 in PBS, pH 7.4. The averaged SEC-SAXS low-resolution model obtained from DAMMIF is extended and asymmetric with one prominent protrusion located on one of its ends, and a bulge on its other end. A representative structure prediction model of BC120 is overlaid on the SEC-SAXS envelope. The backbone nucleotides G14, G15, C16, G34, C35, U36 are highlighted in yellow. These data are rotated 90° from the top conformation. The scale bar represents D_max obtained from the P(r) analysis. (B) Same as (A) with BC120 G25C. The G25C mutation is highlighted with the C25 backbone in red. (C) Same as (A) with EB120 in PBS, pH 7.4. The low-resolution SEC-SAXS envelope of EB120 is y-axis rotationally symmetric and has two prominent protrusions located on its ends. A representative structure prediction model of EB120 is overlaid on the SEC-SAXS envelope. The backbone nucleotides G14, G15, C16, A34, C35, A36 are highlighted in yellow and are predicted to form the hydrogen bonds of the pseudoknot. (D) Magnification of the first ∼45 nt of the representative SimRNA structure prediction model of BC120 forming a pseudoknot with two stem–loops. The stem–loop nearest to the 5′ end is labeled a, while the following downstream stem–loop is labeled b. Nucleotide chemical structures are highlighted in yellow as in (A) and demonstrate the predicted hydrogen bond interactions in the pseudoknot. (E) Same as (D) with BC120 G25C where the C25 backbone is highlighted in red. (F) Same as (D) with EB120 demonstrating two independent stem–loops. Nucleotide chemical structures are highlighted in yellow as in (C). (G) Secondary structure of the first ∼45 nt of BC120 derived from the predicted tertiary structure model using RNApdbee 2.0 (Zok et al. 2018). Hydrogen bonds highlighted in red demonstrate the model constraints used. Circles shaded in yellow behind nucleotides indicate the highlighted nucleotides in (D). (H) Same as (G) with BC120 G25C. Circle in red behind C25 is highlighting the change compared to BC120. (I) Same as (G) with EB120. To be noted, G1–C46, G2–A45, and C3–G44 were also used as constraints but generated a fatal energy structure and were subsequently ignored in the model prediction. Circles shaded in yellow behind nucleotides indicate the highlighted nucleotides in (F).

BC200 Alu domain and its U-turn mutant have the canonical Alu RNA fold that EB120 lacks

Since the U-turn motif nucleotide triad is thought to preserve the 5′ pseudoknot domain within the first ∼50 nt of the RNA, we sought to evaluate whether BC120, BC120 G25C, and EB120 adopted this structure (Skryabin et al. 1998; Weichenrieder et al. 2000; Huck et al. 2004; Ahl et al. 2015). Using SimRNA v3.20 with a minimal number of secondary structure constraints, 20,000,000 atomistic models were calculated for all three RNAs (Labuda and Striker 1989; Weichenrieder et al. 2001; Booy et al. 2016). SimRNA clustering was conducted based on the energy-based lowest 1% of all trajectories. Representative tertiary models were superimposed on the SEC-SAXS DAMFILT envelopes using DAMSUP, generating 4000 high-resolution models. When superimposed, NSD ranges for the top five models were obtained for BC120 (0.809–0.844), BC120 G25C (0.845–0.869), and EB120 (0.894–0.924). Overall, the top five models generated fit the SEC-SAXS envelopes well.

A single representative model for BC120, BC120 G25C, and EB120 was overlaid with the respective SEC-SAXS envelope (Fig. 7A–C), while other models were observed to have similar structures (Supplemental Figs. S10–S12). The first ∼45 nt of BC120 and BC120 G25C modeled two stem–loops, which interact with one another to form a pseudoknot domain consistent with the high-resolution structure determined for 7SL (Fig. 7D,E; Weichenrieder et al. 2000). On the other hand, the first ∼45 nt of EB120 modeled two stem–loops that did not form a pseudoknot domain (Fig. 7F). Secondary structure mapping of the BC120 and BC120 G25C predicted SimRNA structure models demonstrate two stem–loops in the first ∼50 nt as Alu structures canonically possess (Fig. 7G,H; Zwieb et al. 1996). The EB120 secondary structure mapping demonstrates a noncanonical representation, where the U-turn motif is absent (Fig. 7I). We also predicted the structure of BC120, BC120 G25C, and EB120 using AlphaFold 3 (Abramson et al. 2024). The deep neural network predicted BC120 and BC120 G25C to have near identical structures containing a pseudoknot domain (Supplemental Figs. S13, S14), while EB120 was structurally unique and lacked a pseudoknot domain (Supplemental Fig. S15). Both BC120 and BC120 G25C are modeled similarly using either SimRNA or AlphaFold 3, while EB120 has significantly different models (Supplemental Fig. S16). Using a combination of in vitro and in silico methods, as well as only in silico approaches, it is evident that BC120 and BC120 G25C possess the canonical Alu RNA fold, while EB120 lacks it.

DISCUSSION

Over 1 million copies of human Alu elements exist, with 99% of the elements being unique in sequence (Lander et al. 2001; Umylny et al. 2007). The more ancient Alu elements have an accumulation of mutations, which inevitably would impact the conservation of their U-turn motifs (Bennett et al. 2008). Study of these Alu RNAs with noncanonical U-turn nucleotide triads is challenging due to the short length, low abundance, and homology of RNA polymerase III transcribed noncoding Alu RNA. Detection techniques are often limited to northern blots or primer-based amplifications, and these methods are prone to cross-reaction with abundant 7SL or BC200, which mask the identity and quantity of the less abundant Alu RNA (Liu et al. 1994; Booy et al. 2024). Furthermore, identifying novel RNA polymerase III transcribed noncoding Alu RNA has commonly relied on next generation sequencing data sets (Conti et al. 2015). This results in a small number of identified unique RNA as a major limitation is the low abundance of Alu RNA, and the challenges in uniquely identifying the highly homologous transcripts, especially when accounting for error rates associated with the technique. The methodology used to detect BC120 was effective in specifically identifying a unique Alu RNA (Booy et al. 2024).

The investigation of EB120 is also an example of the simplest, biologically expressed human RNA polymerase III transcribed noncoding Alu RNA; an Alu RNA that is mapped back to a left arm Alu monomer not embedded within an annotated gene (Fig. 2). Its difference in its A-box relative to other Alu RNA may also explain its reduced abundance and cell specificity (Englert et al. 2004; Kim et al. 2017), while maintaining similar cellular localization (Fig. 3; Kondrashov et al. 2005; Lin et al. 2008; Akopian et al. 2013; Jang et al. 2017; Booy et al. 2021, 2024; Gussakovsky et al. 2023). Another plausible explanation is the requirement of SRP9/SRP14 interaction in order for the heterodimer to promote transcription as observed with 7SL and BC200 (Gussakovsky et al. 2023). As EB120 is an AluJo-derived RNA, our result of nonassociation with SRP9/SRP14 is consistent with more ancient Alu elements having an accumulation of mutations that inhibit SRP9/SRP14 interaction (Fig. 4; Bennett et al. 2008). This suggests that there are likely thousands of expressed RNA polymerase III transcribed noncoding Alu RNAs that are conformationally similar to EB120 but may be expressed in specific cell types or during specific stresses.

The U-turn mutant having a different migration pattern compared to the wild type indicates a subtle difference in shape or charge exposure (Fig. 1B,C). We observe steady or increased levels of SRP9/SRP14 to the U-turn motif mutants in the high-concentration streptavidin pulldowns (Fig. 1D), standing in contrast to other experimental designs investigating binding affinity (Chang et al. 1997). Our experiment used high concentrations of RNA, as well as human cell–derived SRP9/SRP14, suggesting subtle differences exist and need to be accounted for in the interpretation of experiments investigating the steady-state associations between Alu RNA and SRP9/SRP14. Our results demonstrating the U-turn motif mutant causing a reduction in expression are consistent with the mutation directly impacting SRP9/SRP14 downstream biological processes (Fig. 1E,F; He et al. 1994; Bennett et al. 2008; Ivanova et al. 2015), such as plausibly impacting RNA turnover in a cotranscriptional manner (Gussakovsky et al. 2023). The higher melting temperature of BC120 G25C relative to BC120 is another indicator of the impact of the mutation on the stability of the RNA (Fig. 5A). SRP9/SRP14 can induce a stable RNA structure through its interaction (Weichenrieder et al. 2000). Consequently, this could conceivably force the unique native conformations to a single state upon interaction. It is plausible that during perturbations such as electric current, thermal denaturation, or association with SRP9/SRP14, that differences between the wild-type and U-turn mutant would be discernible.

Our results support the concept of the steady-state BC200 Alu domain and its U-turn mutant having similar or subtly different conformations (Figs. 5–7). The similarity in shape and size of EB120 relative to the BC200 Alu domain demonstrates the resolution that is required to determine the conformational differences between Alu RNA. Overall, the data are self-consistent (Table 1) and comparable with other Alu RNA biophysical analyses. The sense LincRNA-p21 Alu RNA is a dimeric Alu RNA, with s°_20°C,w 5.56 S (compared to BC200 Alu domain ∼4.5 S), R_H of 7.27 nm (∼3 nm), D_max of 18.5 nm (∼14 nm), and R_G of 6.09 nm (∼4 nm) (D'Souza et al. 2022). The 118 nt truncation of the Alu domain of the Plasmodium falciparum 7SL (PfAlu118) was also assessed by SAXS. PfAlu118 lacks a pseudoknot yet interacts with SRP9/SRP14 through its open Y-shaped conformation formed by its first ∼80 nt (Soni et al. 2021). It has comparable D_max of 11.8 nm and R_G of 3.33 nm to the BC200 Alu domain, but its reported conformation is significantly different, likely due to the additional nucleotides that form the pseudoknot.

Investigations into Alu RNA structural diversity has been limited to analyses of the Alu domain of 7SL in various organisms, consensus Alu sequences, or Alu RNAs that are part of a larger RNA (Strub et al. 1999; Bousset et al. 2014; Kempf et al. 2014; Ahl et al. 2015; Soni et al. 2021; D'Souza et al. 2022). The canonical Alu RNA fold and its necessity for SRP9/SRP14 interaction was first demonstrated by 7SL and similarly shown with the AluJ consensus (Weichenrieder et al. 2000; Ahl et al. 2015). Our data demonstrating the BC200 Alu domain possessing this fold is consistent with previous studies indicating a strong binding affinity between the BC200 Alu domain and SRP9/SRP14 (Fig. 7; Supplemental Figs. S13, S14; Booy et al. 2018, 2024; Gussakovsky et al. 2023). The lack of difference in structural modeling between the wild type and U-turn mutant is likely due to subtle differences. A critical triad of nucleotides near the SRP9/SRP14 binding site of 7SL are the nucleotides G4, U23, and G24 (corresponding to G5, U24, and G25 in the BC200 Alu domain, Fig. 1A) (Weichenrieder et al. 2000). G4 and U23 base pair and position G4 to base stack with G24. Under a G24C mutation in 7SL (He et al. 1994; Chang et al. 1997), and a G25C mutation in additional Alu RNA (Bennett et al. 2008; Ivanova et al. 2015) there is a loss of SRP9/SRP14 association, which is consistent with the mutation minimizing base stacking of G4. In the context of BC200, we anticipate there will be a subtle difference in structure caused by the U-turn guanosine mutation, and this will likely impact affinity to SRP9/SRP14 in cells. Existing high-resolution structures of SRP9/SRP14 in complex with Alu RNAs indicate the presence of numerous electrostatic interactions along the binding interface (Weichenrieder et al. 2000; Bousset et al. 2014; Ahl et al. 2015). As such, it is plausible that subtle structural differences within the U-turn motif could reduce the alignment of the RNA backbone phosphate groups with the heterodimer amino acid side chains involved in these interactions, thereby lowering the overall binding affinity. On the other hand, this mutation is likely insufficient for the breaking of hydrogen bonds formed between the two stem–loops that form the pseudoknot. To verify the distinct differences caused by the U-turn mutant, higher resolution techniques would need to be used.

Structural modeling implies EB120 lacks a canonical Alu RNA fold (Fig. 7; Supplemental Fig. S15). This result is consistent with the primary sequence suggesting that the nucleotides present in the stem–loops are not compatible for pseudoknot formation (GGC–ACA in EB120 compared to GGC – GCU in BC200) (Fig. 2F). The primary sequence also demonstrates that in the critical SRP9/SRP14 interaction nucleotide triad (U4, U24, G25 in EB120), there is no base pair partner for U24. This is consistent with our data indicating that SRP9/SRP14 does not interact with EB120, even at high concentrations (Fig. 4). This raises the question of whether the U-turn motif nucleotide triad, in addition to compatible base-pairing nucleotides on the stem–loops, is required for pseudoknot formation and SRP9/SRP14 interaction. As more biologically expressed Alu RNAs are investigated, it would be beneficial to correlate SRP9/SRP14 binding with the presence of the U-turn motif nucleotide triad and base-pairing partners across the two stem–loops.

Alu RNA functions are commonly thought to be regulated by SRP9/SRP14 (Gussakovsky et al. 2024). The expression of EB120 with no direct interaction with SRP9/SRP14 provokes consideration of the diversity of Alu RNA functions. Some functions that Alu RNA can perform only rely on sequence complementarity, and not specific structures. This includes Alu RNAs that function as miRNA sponges, or are parts of inverted Alu repeats on mRNA (GLI1) or long noncoding RNA (LincRNA-p21), driving A-to-I editing events, or localization, respectively (Chillón and Pyle 2016; Lazzari et al. 2017; Di Ruocco et al. 2018). Until now, investigations into noncoding Alu RNA function have been skewed to the highest expressed, and best studied cases of 7SL, BC200, and BC120 (Gussakovsky and McKenna 2021; Booy et al. 2024). EB120 is likely only one of many Alu RNAs which have noncanonical Alu RNA structures, and further studies into non-SRP9/SRP14 mediated function are necessary to fully comprehend the general functions of Alu RNA. Overall, our study demonstrates an alteration in BC200 expression upon introduction of a U-turn motif mutation, and the BC200 Alu domain possessing a canonical Alu RNA conformation. We also describe the identification of a novel RNA polymerase III transcribed noncoding human Alu RNA called EB120 that lacks the canonical Alu RNA fold. Taken together, our results suggest a more diverse investigation into different Alu RNA would provide novel insights into general Alu RNA function.

MATERIALS AND METHODS

Cell culture and reagents

The MCF-7, MDA-MB-231, SK-BR-3, T-47D, and HeLa cell lines were a gift from Dr. Spencer Gibson. The SK-OV-3, and A549 cell lines were a gift from Dr. Peter Pelka. The HEK293T cell line was a gift from Dr. Thomas Klonisch, the A375 cell line was a gift from Dr. Jens Kurreck, and the A2780 cell line was a gift from Dr. Benjamin Tsang. Cell culture conditions were performed as previously published (Booy et al. 2016). All DNA primers and TaqMan probes were purchased from Integrated DNA Technologies. T4 RNA Ligase 1 was purchased from New England BioLabs. All standard laboratory chemicals and reagents were purchased from Thermo Fisher Scientific.

In vitro transcription and RT-qPCR

In vitro transcription and purification by gel filtration chromatography of BC120, BC120 G25C, and EB120 was performed as described previously (Booy et al. 2012b). Ten millimolars of MgCl₂ were required for efficient transcription and to ensure long-range interactions within Alu RNA were maintained (Ahl et al. 2015). Real-time quantitative PCR (RT-qPCR) of BC200 and EB120 was performed using an Applied Biosystems StepOnePlus instrument (Thermo Fisher Scientific) with the iTaq Universal Probes One-Step kit (Bio-Rad), according to the manufacturer's protocol. TaqMan probes possess a 5′ FAM label, internal ZEN quencher (nine bases from the 5′ fluorophore), and a 3′ Iowa Black fluorescent quencher. Primer concentrations were maintained at 500 nM and probe concentrations at 250 nM for all assays. RT-qPCR of other RNA was performed with the iTaq Universal SYBR Green One-Step kit (Bio-Rad), according to the manufacturer's protocol. To prepare a standard curve, serial dilutions of the EB120 were made in RNase-free water containing 5 ng/µL murine carrier RNA. A total of 25 ng RNA was used as template for every reaction. Reaction specificity was confirmed by agarose gel electrophoresis of reaction products and cloning and Sanger sequencing of reaction products. Primer/TaqMan probe set used for EB120 includes EB120 +4 to +20 Forward, EB120 Probe, and EB120 +96 to +114 Reverse, while for BC200 includes BC200 Forward, BC200 Probe, and BC200 Reverse. Sequences for all primers and probes used for RT-qPCR reactions are listed in Supplemental Table S1.

Cell lysis, RNA purification, and RNA quantification

Cytoplasmic extracts were prepared by lysing cell pellets with 25 mM HEPES, pH 7.9, 5 mM KCl, 0.5 mM MgCl₂, 0.5% (v/v) NP-40 (cytoplasmic lysis buffer) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), and RiboLock RNase inhibitor (Thermo Fisher Scientific) followed by 10 min of end-over-end mixing at 4°C. The lysates were then adjusted to a composition of 25 mM HEPES, pH 7.9, 5 mM KCl, 0.5 mM MgCl₂, 0.25% (v/v) NP-40, 100 mM NaCl (IP buffer). Insoluble material was removed by centrifugation at 2500g at 4°C for 5 min, and supernatants were collected. Cytoplasmic extracts and whole-cell lysates were obtained with the GeneJET RNA Concentration and Cleanup Micro Kit (Thermo Fisher Scientific), according to the manufacturer's protocol. Subcellular fractionation was performed with the Subcellular Fraction Kit for Cultured Cells (Thermo Fisher Scientific), according to the manufacturer's protocol, with substitution of the micrococcal nuclease digestion by solubilizing the pellet with binding buffer from the GeneJET RNA Concentration and Cleanup Micro Kit. RNA isolated from normal human tissue was purchased from Takara Bio. Isolated and purchased RNA was quantified using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).

Streptavidin pulldown

In vitro transcribed BC120, BC120 G25C, and EB120 were ligated with pCp-biotin (Jena Bioscience) to affix a biotin label on the 3′ end of the RNA with T4 RNA Ligase 1. The 100 µL ligation reaction consisted of 1× NEB T4 RNA ligase buffer, 1 mM ATP, 30 µM pCp-biotin, 100 units T4 RNA ligase 1, 10% DMSO, 10% PEG 8000, 40 units RiboLock RNase inhibitor with 3 µM RNA. The ligation mixture was incubated at 12°C for 3 h prior to purification with the GeneJET RNA Concentration and Cleanup Micro Kit, according to the manufacturer's protocol. Biotinylation efficiency was assessed by performing a slot blot on a positively charged nylon membrane, and detection was performed with streptavidin-HRP (S2438, Sigma-Aldrich). Streptavidin pulldowns were performed with cells that were ∼80% confluent after 24 h, with seeding at a cell density of 1.5 × 10⁷ HEK293T or 1.1 × 10⁷ SK-BR-3 cells per 150 mm tissue culture dish. Cytoplasmic lysates were collected and adjusted to IP buffer as described above. Protein content was measured with a standard Bradford assay and normalized to a concentration of 5.0 mg/mL (HEK293T) or 2.5 mg/mL (SK-BR-3). A total of 1500 µg (HEK293T) or 750 µg (SK-BR-3) of cytoplasmic lysate and 250 nM of biotinylated RNA were mixed end-over-end for 1 h at 4°C. Forty microliters of pre-equilibrated Pierce streptavidin magnetic beads (Thermo Fisher Scientific) were added to the lysate and mixed end-over-end at 4°C for 1 h. Following incubation, beads were subjected to four washes of IP buffer. Magnetic beads were then pelleted, supernatant discarded, and eluted with 1× SDS load dye for SDS-PAGE and western blotting.

SDS/PAGE, western blotting, and antibodies

Protein isolation from cultured cells was conducted by lysing the cells as described above. Protein quantification was performed by a standard Bradford assay. SDS/PAGE and western blotting were performed as previously described (Booy et al. 2012a). The Rabbit anti-SRP9 (11195-1-AP) and Rabbit anti-SRP14 (11528-1-AP) antibodies were purchased from Proteintech. The Mouse anti-GAPDH (AM4300) and Rabbit anti-ACTB (PA1-183) antibodies were purchased from Thermo Fisher Scientific. The negative control antibody raised against Protein I from Neisseria gonorrhoeae (isotype control) (MABC004) was purchased from Sigma-Aldrich.

Plasmid construction and transfection

Plasmids transfected were either an empty pcDNA3.1 Hygro(+) vector (Empty), BC200 (+1 to +201) (BC200), BC200 with a G25C mutation (+1 to +201) (BC200 G25C) or EB120 (+1 to +121) (EB120) cloned using the BglII and MluI restrictions on the pcDNA3.1 Hygro(+) vector. To be noted, the G25C mutation does not overlap with the A-box of BC200 and is therefore not expected to impact RNA polymerase III transcription initiation. Plasmids were transfected using polyethylenimine (PEI) Max (Polysciences Inc.), according to the manufacturer's protocol. Reverse transfections were performed by combining 4 µg of plasmid with 6 µg of PEI Max in 400 µL Opti-MEM media per well of a 6-well plate.

RNA thermal denaturation

BC120, BC120 G25C, and EB120 in PBS, pH 7.4 were diluted to 5 µM and heated at 95°C for 3 min prior to gradual cooling to 4°C and equilibration at room temperature. The RNAs were then diluted further with PBS, pH 7.4 to 1 µM and added to a 1 cm quartz cuvette with a volume of 1500 µL for analysis in an Evolution One UV-Vis spectrophotometer (Thermo Fisher Scientific) equipped with a single-cell Peltier system for temperature control (Thermo Fisher Scientific). Sample temperature was increased from 22°C to 95°C at a rate of 2°C/min. Absorbance at 260 nm was measured every 10 sec with a bandwidth of 1 nm. Plotted data originated from the fit of a sigmoidal curve and its measured first derivative. Using the thermal denaturation data, the hyperchromicity effect was corrected for in the calculation of RNA concentrations (Puglisi and Tinoco 1989).

Spectropolarimetry

BC120, BC120 G25C, and EB120 RNA in PBS, pH 7.4 were diluted to 5 µM and heated at 95°C for 3 min prior to gradual cooling to 4°C and equilibration at room temperature. Circular dichroism spectra were collected at room temperature on a J-1500 spectropolarimeter (JASCO) in a 1 cm quartz cuvette with a volume of 300 µL. Spectra were acquired in the range between 200 and 320 nm at a rate of 100 nm/min with 2 sec response, bandwidth of 1 nm, and averaged over three measurements with a buffer baseline subtraction. The observed ellipticity was normalized using Δε = θ/(32,980 × c × l), where Δε is the difference in the molar absorption of the right- and left-handed circularly polarized light in M⁻¹ cm⁻¹, θ is the ellipticity in m°, c is the concentration of the RNA in mol/L, and l is the path length of the cuvette in cm.

Sedimentation velocity

Sedimentation velocity profiles were measured on a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) using an An-50 Ti 8-place rotor, speed of 30,000 rpm, and temperature of 20°C, as described previously (Meier et al. 2018). Samples were solubilized in PBS, pH 7.4, and concentrations ranged from 450 to 1100 nM. Four hundred microliters of sample and buffer were loaded into the respective channel of the double-sector centerpiece. Radial scans were collected in 10 min intervals for 24 h at 260 nm using an absorption optical system. Data were analyzed using the SEDFIT program to obtain sedimentation coefficients s and frictional ratios f_r = f/f₀ at each concentration (Brown and Schuck 2006). Two-dimensional c(s,f_r) distributions were calculated along 20 evenly spaced points along f_r and 50 variably spaced points along s to demonstrate the populations of all particles in solution. Between 0 S and 1 S, the distance between the points increased by 0.25 S, while between 1 S and 10 S, the distance increased by 0.2 S. Since both sedimentation and diffusion constants were obtained, the distributions were also converted to molecular mass distributions c(s,M). In addition to c(s) distributions, the c(s,f_r) distributions were collapsed along the f_r axis resulting in one-dimensional c(s,*) distributions, accounting for different species possessing different f_r values. We calculated the apparent s, and apparent diffusion coefficient D and measured uncertainties at each sample concentration by fitting these parameters to the combined Lamm and Svedberg equations using the Species Analysis model within SEDPHAT (Schuck 1998, 2003). The obtained values were extrapolated to an infinite dilution and converted to standard conditions (pure water at 20°C) with a buffer density 1.00575 g/cm³, buffer viscosity 0.010194 P, and partial specific volumes of 0.694 ± 0.033 cm³/g for BC120, 0.578 ± 0.015 cm³/g for BC120 G25C, and 0.586 ± 0.096 cm³/g for EB120 to obtain s°_20°C,w and R°_H. Figures were prepared using the GUSSI software (Brautigam 2015).

Dynamic light scattering

Dynamic light scattering data were collected using a Prometheus Panta (NanoTemper Technologies). BC120, BC120 G25C, and EB120 in PBS, pH 7.4 with concentrations ranging from 9 to 25 µM were centrifuged at 10,000g for 10 min at 4°C prior to analysis. Standard Prometheus capillaries (PR-C002) were used for analysis. A total of 10 acquisitions of 5000 msec each were conducted at 25°C with 100% DLS power.

Size-exclusion chromatography coupled small-angle X-ray scattering (SEC-SAXS)

Data were obtained through SEC coupled small-angle X-ray scattering (SEC-SAXS) at the B21 BioSAXS beamline located at the Diamond Light Source. We used an Agilent 1200 HPLC equipped with a specialized flow cell, which has been described previously (Meier et al. 2018). The RNAs were concentrated to 3 mg/mL using PBS, pH 7.4 and loaded on a pre-equilibrated Shodex 403-4F size exclusion chromatography column. Scattering data were analyzed using ATSAS suite v2.8 and Chromixs to correct the baseline samples’ peak intensity using the buffer as reference (Franke et al. 2017; Panjkovich and Svergun 2018). Guinier analysis [q² vs. ln(I(q))] was used to determine the samples’ quality and to calculate the radius of gyration (R_G) on the inverted space (Putnam 2016). The RNA folding profile was assessed by a dimensionless Kratky plot qR_G versus $q{R}_G^2\times \left(I\left(q\right)/I\left(0\right)\right)$ (Burke and Butcher 2012). We then used a paired distribution function [P(r)] to determine the real-space R_G and the maximum particle dimension (D_max) using GNOM (Svergun 1992). The information of the P(r) plot was used as input data for DAMMIN (Franke and Svergun 2009). DAMMIN generated models in each condition by using different seeds for each model while ensuring symmetry with equal parameters. An average model was produced using DAMAVER, and DAMFILT was used to create a filtered representative structure model (Volkov and Svergun 2003). The hydrodynamic properties were calculated for each model using the program HYDROPRO to validate the models (Ortega et al. 2011).

Computational RNA structure determination

Tertiary structures were generated using SimRNA v3.20 using a minimal number of secondary structure constraints shared by the high-resolution structure of 7SL, conservation of nucleotides in Alu elements, and secondary structure determination in BC200 (Labuda and Striker 1989; Weichenrieder et al. 2001; Booy et al. 2016). SimRNA v3.20 is a software application that uses a simplified model for RNA structure, and it functions as a Monte Carlo sampler (Boniecki et al. 2015). To generate high-resolution structures, we performed 20 million SimRNA iterations in replica exchange mode. The process of SimRNA clustering was executed on the energy-based lowest 1% of all trajectories. Representative tertiary structures were fitted using SAXS DAMFILT structures. These tertiary structures were created computationally, and they comprised 4000 high-resolution models. The high-resolution structures were fit within the SAXS envelopes using DAMSUP and measured based on the normalized spatial discrepancy (NSD). The models chosen exhibited an NSD value below 1.00, indicating a close fit with the SAXS model. Using Chimera, models with the best fit were superimposed on SAXS structures to represent these models. Secondary structure modeling of the tertiary structure models was generated by RNApdbee 2.0 under default settings (Zok et al. 2018). AlphaFold 3 structure predictions were run under default settings (Abramson et al. 2024).

Sequence alignment of Alu elements

The consensus AluJo element was described as previously published (Price et al. 2004). All 43,903 human AluJo elements were obtained from the RepeatMasker database (Bao et al. 2015). All AluJo elements with a length of 117 nt were collected (786 in total), oriented in the sense direction, and aligned using Muscle 3.8.425 (Edgar 2004). Alignment is provided in Supplemental Table S2. Proportion aligned to consensus was calculated as the total proportion of nucleotides of individual AluJo elements, which aligned to the AluJo element consensus sequence.

Rapid amplification of cDNA ends (RACE)

MCF-7 or brain tissue RNA was ligated with a 5′ phosphorylated 3′ DNA adapter (Modban) with T4 RNA Ligase 1. The 100 µL ligation reaction consisted of 1× NEB T4 RNA ligase buffer, 1 mM ATP, 1 µM Modban, 100 units T4 RNA ligase 1, 10% DMSO, 10% PEG 8000, 40 units RiboLock RNase inhibitor with 3 µg RNA. The ligation mixture was incubated at 12°C for 3 h prior to purification with the GeneJET RNA Concentration and Cleanup Micro Kit according to the manufacturer's protocol. These RNAs were then reverse transcribed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer's protocol with slight modifications. The reaction consisted of 2 pmol of a primer complementary to the 3′ adapter (Ban1), and 20 pmol of a strand switching primer (SSP). The SSP primer was used to add a 5′ adapter by exploiting the addition of three to four cytosines to the cDNA in the process of reverse transcription by the Moloney murine leukemia virus (MMLV) reverse transcriptase (Zhu et al. 2001). The reaction was incubated at 55°C for 90 min, prior to inactivation at 85°C for 5 min. The generated cDNA was used as template for both 5′ and 3′ RACE, and internal primers were designed to be specific to the AluJo element found at Chr1: 116709982–116710098 (GRCh38/hg38). For 5′ RACE, nested PCR was used with a primer complementary to the 5′ adapter (SSP PCR Forward) with two specific internal reverse primers (EB120 +96 to +114 Reverse followed by EB120 +44 to +68 Reverse). For 3′ RACE, nested PCR was used with a primer complementary to the 3′ adapter (Ban1) with two specific internal forward primers (EB120 +4 to +20 Forward followed by EB120 +44 to +68 Forward). PCR products were subsequently cloned, and Sanger sequenced to confirm the identity sequence of the transcript. The list of primers and oligonucleotides used for RACE is provided in Supplemental Table S1.

RNA coimmunoprecipitation

RNA coimmunoprecipitations were performed with SK-BR-3 cells that were ∼80% confluent after 24 h. To achieve this, for native and formaldehyde RNA coimmunoprecipitation, SK-BR-3 cells were seeded at a cell density of 1.1 × 10⁷ cells per 150 mm tissue culture dish. For EB120 overexpression, SK-BR-3 cells were seeded at a cell density of 1.6 × 10⁷ cells per 150 mm tissue culture dish. Two 150 mm tissue culture dishes were used per condition analyzed. For native and EB120 overexpression RNA immunoprecipitations, SK-BR-3 cells were lysed with cytoplasmic lysis buffer (described above) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail, and RiboLock RNase inhibitor followed by 10 min of end-over-end mixing at 4°C. The lysates were then adjusted to IP buffer (described above) prior to insoluble material being removed by centrifugation at 2500g at 4°C for 5 min, and collection of supernatants. For formaldehyde RNA immunoprecipitation, SK-BR-3 cells were cross-linked with 0.1% formaldehyde in PBS with rocking for 14 min. Formaldehyde was quenched with a final concentration of 125 mM glycine with rocking for 5 min. Cells were then scraped in cold PBS containing 1% bovine serum albumin and consolidated prior to centrifugation at 1000g at 4°C for 5 min. These pellets were washed with PBS three times prior to lysis with 1 mL of 50 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.1% (v/v) SDS, 0.5% (v/v) sodium deoxycholate (RIPA buffer) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail, and RiboLock RNase inhibitor. Samples were sonicated with five cycles of 10 sec on and 10 sec off with 30% output (Branson Ultrasonics Sonifier SFX150 Cell Disruptor, Thermo Fisher Scientific). Cell debris was pelleted by centrifugation at 21,000g at 4°C for 10 min.

After collection of lysates, protein content was measured with a standard Bradford assay and normalized to a concentration of 2.5 mg/mL. Antibodies used included anti-SRP9 (11195-1-AP) (4 µg), anti-SRP14 (11528-1-AP) (8 µg), and isotype control (MABC004) (3 µg). Antibodies were mixed with a total of 750 µg of lysate for 1 h with end-over-end mixing at 4°C. After antibody incubation, 40 µL of pre-equilibrated Pierce protein A/G magnetic beads (Thermo Fisher Scientific) were added to the lysate and mixed end-over-end at 4°C for 1 h. Following incubation, beads were subjected to four washes of IP buffer for native and EB120 overexpression coimmunoprecipitations and four washes of RIPA buffer for formaldehyde RNA coimmunoprecipitation. For native and EB120 overexpression coimmunoprecipitations, RNA from each sample was then purified using GeneJET RNA Concentration and Cleanup Micro Kit according to the manufacturer's protocol. For formaldehyde RNA coimmunoprecipitations, samples were resuspended in reverse cross-linking buffer (1× PBS, 2% N-lauroyl sarcosine, 10 mM EDTA, 5 mM DTT [add fresh]) and 100 µg proteinase K (Thermo Fisher Scientific) and then incubated for 1 h at 42°C, followed by 1 h at 55°C, prior to purification with the GeneJET RNA Concentration and Cleanup Micro Kit. For EB120 overexpression RNA coimmunoprecipitations, purified nucleic acids were additionally treated with DNase I (Thermo Fisher Scientific) according to the manufacturer's protocol, with incubation at 37°C for 1 h prior to purification with the GeneJET RNA Concentration and Cleanup Micro Kit. Coimmunoprecipitated RNA was quantified with RT-qPCR using 5% of each immunoprecipitation as template for every reaction. BC200 and EB120 levels were measured by RT-qPCR as described above as the mean of three technical replicates, and are representative of three biological replicates with error bars indicating ±standard deviation.

DATA DEPOSITION

SAXS data have been deposited to SASDB under accession numbers SASDU59 for BC120, SASDWR3 for BC120 G25C, and SASDU69 for EB120.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.