Two microPeptides are translated from a KSHV polycistronic RNA in human cells by leaky scanning mechanism

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a 3.0 kb polyadenylated RNA (T3.0) in the opposite strand of the open reading frame 50 (RTA) gene. The T3.0 was mis-annotated as a noncoding RNA but found to be associated with ribosomes and carries at least four translatable sORFs. Two of them, namely vSP-1 and vSP-2, have been characterized. vSP-1 enhances RTA expression by blocking RTA self-ubiquitylation and proteasome-associated degradation. T3.0 RNA is a polycistronic RNA. Furthermore, polycistronic translation has been observed in most of the cases of small peptides (microPeptides) translated from previously annotated noncoding RNAs in eukaryotes. In an effort to elucidate the mechanism underlying polycistronic sORF translation in eukaryotic cells, we found that T3.0 RNA translates vSP-1 and vSP-2 through a leaky scanning mechanism.

1. Introduction

Noncoding RNAs (ncRNAs) have emerged as a new and crucial layer of biological regulation, playing roles in diverse cellular processes. However, some ncRNAs contain small open reading frames (sORFs) that can be bona fide translated into functional small peptides, also designated microPeptides [1–3]. The sORFs from which micropeptides are translated can be encoded in 5’ UTRs or polycistronic mRNAs, but more found in originally mis-annotated ncRNAs [4]. Such sORF-containing ncRNAs and microPeptides are also identified in viruses [2,5,6].

sORFs in ncRNAs (mis-designated) are commonly polycistronic as several small peptides are synthesized from the single RNA. Given that a polycistronic mRNA giving rise to multiple peptides from a single transcript is a prokaryotic translation mechanism, and usually does not occur in eukaryotic cells, polycistronic sORFs represent a novel translation mode in eukaryotic cells. In eukaryotic cells, a ribosome enters at the 5’ end of a eukaryotic mRNA by recognizing the m7G cap, scans the RNA until the first AUG codon is reached and translation initiation occurs [7]. Although polycistronic mRNAs are often seen in viral genomes, only the first open reading frame is translated for each mRNA except a few cases where IRES (internal ribosome entry site) is used [8–11]. Thus, it appears that there is a unique and novel translation mode for eukaryotic sORF synthesis and elucidation of the novel translation mechanism is vital to understand the regulation of microPeptide expression and biological significance in eukaryotic cells.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus etiologically associated with Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease [12]. Like all herpesviruses, KSHV has two life cycles, latency and lytic replication. The switch of KSHV between latency and lytic replication is controlled by a virally encoded transcriptional activator, namely, RTA (replication and transcription activator), which is necessary and sufficient to disrupt latency and initiate viral lytic replication [13]. During KSHV reactivation, a 3.0-kb RNA, designated T3.0 is transcribed from the opposite strand of RTA gene [14]. Although T3.0 was originally annotated as a non-coding RNA, it was found later that T3.0 transcript carries a series of sORFs and some of the sORFs have potential to make microPeptides as the RNA is associated with ribosomes [6]. Two microPeptides, designated vSP-1 and vSP-2, 48- and 27-aa in length respectively, were detected in KSHV-replicating cells [2]. MicroPeptide vSP-1 physically interacts with RTA at the PARS II domain and blocks the self-ubiquitylation of RTA by the internal E3 ligase located at the RING domain, therefore increasing RTA abundance in cells and facilitating KSHV lytic replication [2]. In the current study, we take advantage of T3.0 RNA and its multiple sORFs to study the polycistronic translation mechanism in eukaryotic cell.

2. Materials and Methods

2.1. Cells and transfections

Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and antibiotics (penicillin-streptomycin), and grown at 37ºC under 5% CO₂ in a humidified incubator. 293T cells were plated onto 24-well tissue culture plates 24 h prior to transfection. When reached 60 to 80% confluent, cells were transfected with plasmid DNA alone or in combination with GFP expression vector as a transfection control using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol.

2.2. Plasmid constructs

The psiCHECK-2 (Invitrogen) dual-luciferase construct harbors firefly and Renilla luciferase open reading frames under the promoters of the thymidine kinase (TK) and simian virus 40 (SV40) promoters, respectively. To clone T3.0 fragments in front of firefly luciferase open reading frame, a Spe I site was introduced into the plasmid by amplifying the whole psiCHECK-2 sequence and self-ligating with a pair of primers carrying the SpeI sequence. The psiCHECK-vSP1/2 construct was generated by inserting the PCR-amplified T3.0 fragment (5’UTR-vSP-1-vSP-2) into the SpeI - Bsp1407I site of the backbone in front of the Firefly luciferase open reading frame. A series of point mutants were generated by substituting particular nucleotides (such as mutating the AUG start codon to GUC for the v2-mAUG mutant) using a KOD-Plus-Mutagenesis Kit (Toyobo). The deletion mutations, for example the sv1–v2 construct (shortening the distance between vSP-1 and vSP-2) were generated by PCR amplifying the prototype sequence omitting part of sequence between two ORFs and then self-ligating the plasmids.

For the vSP-GFP constructs, the T3.0 sequence was amplified from the KSHV-BAC36 by PCR and cloned into pCR3.1 vector at EcoRI and XhoI sites. The enhanced green fluorescent protein (EGFP) gene (without AUG) was PCR amplified using the EGFP-N1 plasmid as a template. The EGFP fragment was then cloned into T3.0 construct via three-step sequential overlap extension PCR. For example, to generate the v1-GFP construct (EGFP was inserted and followed after the vSP-1 sequence without TGA), we first generated the 5’UTR-vSP-1-EGFP by overlap extension PCR, then added the subsequent sequence of T3.0 to 5’UTR-vSP-1-EGFP by second overlap extension PCR. Finally, the fragment fused with the EGFP gene was cloned into the pCR3.1 vector at the EcoRI and XhoI sites. The point and deletion mutations were generated in the same way as constructing the dual-luciferase constructs above.

2.3. Dual-luciferase reporter assays

293T cells were seeded onto 24-well plates 24h prior to transfection. When cells reached 60 to 70% confluent, DNA transfections were performed using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Twenty-four hours post-transfection, cells were harvested and lysed in passive lysis buffer (Promega). Firefly and Renilla luciferases activities were measured and quantified with a TriStar Multimode Reader. The value of fold activation represents at least three independent experiments with triplicate replicated samples. Error bars represent the standard deviation between replicates.

2.4. Fluorescence microscopy analysis

293T cells were transfected with GFP plasmids using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Twenty-four hours post-transfection, GFP fluorescence was monitored using a fluorescence microscope (Axio-Observer.Z1, Zeiss) with a 10× objective lens. Fluorescence was detected using the spectral band about 400 nm.

2.5. Western blotting analysis

Cells were lysed on ice for 30 min in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% Triton X-100, 1 mM sodium orthovanadate (Na3VO4), 20 mM sodium pyrophosphate, 100 mM sodium fluoride, 10% glycerol, protease inhibitor cocktail (1 tablet in 50 mL lysis buffer)] and then centrifuged in 12,000 rpm for 10 min. The supernatants were mixed with loading buffer and heated at 95°C for 10 min. Protein concentration was measured using the BCA method (Thermo). Equal amounts of protein (50 μg) were separated by SDS-PAGE and subsequently electro-transferred onto the polyvinylidene di-fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 (PBS-T) for 1h at room temperature. Then the membranes were incubated overnight with mouse anti-GFP monoclonal antibody (1:5000) at 4°C. After washing with PBS-T, the membranes were incubated with 800CW goat anti-mouse IgG antibodies (LI-COR Biosciences) as secondary antibody. An Odyssey system (LI-COR) was used for detection of proteins of interest.

3. Results

3.1. T3.0 is a functional polycistronic RNA where both vSP1 and vSP2 sORFs are translatable.

The T3.0 transcript, which is transcribed from the opposite strand of RTA gene (ORF50), was found to carry a series of sORFs. The most 5’ proximal sORF in T3.0 is known to encode a microPeptide vSP-1 of 48-aa. The second sORF is not translatable. The third sORF encoded the microPeptide vSP-2 of 27-aa. The last ORF encodes ORF49, which is not translated from T3.0 but from a 1.2 kb mRNA that shares the same poly(A) terminus with T3.0 RNA [2,6]. MicroPeptides vSP-1 and vSP-2 have been characterized [2]. To confirm that T3.0 is a polycistronic RNA where multiple sORFs can be translated from the single RNA, we inserted a GFP coding sequence in vSP-1 and vSP-2 sORFs, respective, generating the vSP-1-GFP and vSP-2-GFP chimeric constructs. These constructs allow us to visualize the sORF expression using Western and immunofluorenscence assays. After introduced into 293T cells, both vSP-1-GFP and vSP-2-GFP can detected by Western analysis with an anti-GFP antibody (Fig. 1C) as well as GFP fluorescence under a fluorescence microscope (Fig. 1D). Translations of both chimeric proteins were respectively initiated from the vSP-1 and vSP-2 AUG start codons, as the expression were completely abolished by mutating their AUG initiation codons (Fig. 1B–D).

Fig. 1. T3.0 is a polycistronic mRNA.

(A) Schematic representation of vSP-1 and vSP-2 sORFs in T3.0 transcript (the T3.0 transcription and sORF translation initiation positions are numbered according to the KSHV genome GenBank accession number U75698). (B) Structures of vSP-1- and vSP-2-GFP fusion constructs where an EGFP ORF was fused in-frame with vSP-1 and vSP-2 (without the stop code) in T3.0 gene under a CMV promoter. The mutant vectors have the vSP-1 or vSP-2 start codon mutated from AUG to GUC. (C) The expression of the GFP fusion proteins for each construct was shown by Western blot with an anti-GFP antibody. (D) 293T cells transfected with the GFP chimeric constructs were analyzed under a fluorescence microscope and phase contrast and GFP fluorescence was shown.

3.2. vSP-2 is translated through a leaky scanning mechanism

Although the general concept for eukaryotic gene expression is that an mRNA codes for only one single peptide, the polycistronic structure has been frequently found in mis-annotated noncoding RNAs that code for microPeptides [2,15–18]. It suggests that polycistronic translation is common for microPeptide synthesis in eukaryotic cells, representing a non-canonical eukaryotic translation mechanism. vSP-1 and vSP-2 translation provides a platform to elucidate this novel mechanism for polycistronic sORF translation from mis-annotated noncoding RNAs. To study the translation mechanism of vSP-1 and vSP-2, we adapted a luciferase-reporter system. A 5’ proximate sequence of T3.0, from transcriptional initiation site to vSP-2 sORF, was cloned into the psiCHEK-2 vector, where vSP-2 sORF was fused with a firefly luciferase gene (Fig. 2A). There is also a Renilla luciferase in the construct under the control of an independent promoter to provide an internal control of transfection efficiency, allowing us to monitor the ratio of Firefly luciferase to Renilla luciferase for quantitation of vSP-2 sORF expression. In addition, three mutants were generated that mutated vSP-2 translation start codon AUG to GUC, or deleted the entire vSP-2 sORF, or replaced the vSP-2 sORF with the vSP-1 sORF, respectively. These reporter constructs were introduced into 293T cells and vSP-2 translation efficiency was determined by a dual luciferase assay. Result showed that the vSP-2 sORF could be efficiently translated from its AUG codon as mutation the start codon could completely abolish the translation of vSP-2 sORF (Fig. 2B–C). Furthermore, deletion or replacement of the vSP-2 sORF with the vSP-1 sORF reduced luciferase activity, suggesting vSP-2 has a stronger translation initiation ability in comparison to vSP-1 sORF. This result again confirmed that although positioned as the 3^rd sORF in T3.0, vSP-2 is translatable from T3.0 transcript.

Fig. 2. Elucidation of the mechanism underlying vSP-1 and vSP-2 translation using a dual luciferase system.

(A) Diagram of the dual luciferase construct in which the 5’ proximate sequence of T3.0, containing the native 72-nt 5’ UTR, vSP-1 and vSP-2 sORFs, was cloned into the psiCHEK-2 vector under the TK promoter and the vSP-2 sORF is fused with a firefly luciferase ORF in frame. There is also a Renilla luciferase in the vector under the control of an SV40 promoter. (B) A series of mutants derived from the dual luciferase construct are schematically illustrated in the panel and Firefly luciferase activities were determined by dual luciferase assay 24 h post-transfection. (D) To explore the vSP-1 and vSP-2 translation mechanism, additional mutants were designed that restored the Kozak consensus sequence of the vSP-1 (v1Kozak), mutated the AUG of vSP-1 (v1mAUG), or changed the length of the intercistronic sequence between vSP-1 and vSP-2, respectively. (E) The indicated constructs were transfected into 293T cells and a dual-luciferase assay was performed 24 h post-transfection. All experiment was performed in triplicate. Error bars represent the standard deviations of the results.

Using the vSP-2 luciferase reporter system, we sought to investigate the mechanism of T3.0 RNA polycitronic translation. Initiation of translation in eukaryotes has been proposed to follow a scanning model that a 40S ribosome complex identifies the 5’ cap structure of a mRNA and scans for the first AUG and preferred context (Kozak consensus sequence) to be used as translation start codon. How is vSP-2 translated as the third sORF in T3.0 RNA? Two hypotheses were in our consideration. (i) Survey of sequences of each potential sORF in T3.0 showed that both the first (vSP-1) and second sORFs have weak Kozak context but the third sORF (vSP-2) carries a perfect Kozak sequence (gtcAUGa). Our first hypothesis was that such arrangement allows the first sORF to be translated with some ribosomes initiating at its AUG but most ribosomes continue scanning downstream and initiate translation at the AUG of vSP-2 sORF that has a perfect Kozak consensus sequence. (ii) Translation could be re-initiated at vSP-2 AUG codon after completing vSP-1 synthesis including using an internal ribosome entry sequence (IRES)-like element in the intercistronic space that recruits 40S ribosome to the vSP-2 AUG codon. To investigate the translation mechanism of vSP-2, we generated a series of mutants to the vSP-2 luciferase reporter plasmid in consideration of these two hypotheses above (Fig. 2C). vSP-1 possesses a relatively weak Kozak context surrounding its start codon with pyrimidines in both −3 and +4 nucleotides (cccAUGuuu), raising a possibility that 40S ribosome may not efficiently initiate translation at vSP-1 start codon but continues to slide down until vSP-2 start code with a better Kozak context. We mutated the vSP-1 context sequence to a stronger Kozak consensus sequence by changing the nucleotide at −3 and +4 to purines (accAUGguu). A stronger vSP-1 Kozak context dramatically reduced vSP-2 translation by 7-fold. In contrast, mutation of vSP-1 AUG codon to GUC significantly increased vSP-2 translation efficiency (Fig. 2D–E). Furthermore, we also test the possibility that T3.0 uses re-initiation mechanism to achieve polycistronic translation by shortening and elongating the intercistronic sequence between vSP-1 and vSP-2. It has been shown that re-initiation can be promoted by lengthening the intercistronic sequence, probably it may provide more time for Met-tRNAi to bind to 40S ribosome subunit that remains bound on the RNA [19,20]. We found that shortening the intercistronic sequence between vSP-1 and vSP-2 (from 181 to 34 nt) led to significant increase, while elongation of the sequence (181 to 362 nt) decrease in vSP-2 translation efficiency (Fig. 2D–E). These results support a leaky scanning mechanism for translation of vSP-2 and rule out the possible ribosome re-initiation mechanism.

3.3. Confirmation of the leaky scanning mechanism for vSP-2 translation in protein level

To further confirm the leaky scanning translation of vSP-2, we introduced the above mutations into vSP-1-GFP and vSP-2-GFP chimeric constructs, which (i) restored vSP-1 Kozak consensus sequence; (ii) mutated two ATG codons in the vSP-1 sORF; (iii) shortened the intercistronic space sequence; and (iv) doubled the intercistronic sequence (Fig. 3A). These mutants were transfected into 293T cells and subjected to Western and immunofluorenscence analyses. In agreement with luciferase results, the Western analysis revealed that the vSP-2 translation was significant influenced by vSP-1 translation. The GFP protein and fluorescence levels reduced as the vSP-1 translation was enhanced by restoration of a stronger Kozak sequence. GFP expression and fluorescence was increased as the vSP-1 AUG site was mutated to GUC. The shortening of intercistronic sequence between vSP-1 and vSP-2 (from 181nt to 34nt) led to a great increase of GFP protein and fluorescence level, while doubling the space sequence led to the opposite result (Fig. 3B). These results further confirmed the leaky scanning model for vSP-2 translation. A schematic illustration of the leaky scanning model for vSP-1 and vSP-2 translation is presented in Fig. 4.

Fig. 3. Verification of the leaky scanning mechanism for T3.0 sORFs in protein level.

(A) Schematic representation of the vSP-2-GFP fusion construct and its mutants. (B) The indicated constructs were transfected into 293T cells and harvested 48h post-transfection. Whole cell lysates were prepared and subjected to Western blotting with an anti-GFP antibody and quantitated (C). (D) 293T cells transfected with the GFP chimeric constructs were analyzed under a fluorescence microscope and phase contrast and GFP fluorescence was shown.

Fig. 4. A model of vSP-1 and vSP-2 synthesis from KSHV T3.0 polycistronic mRNA.

40S ribosome recognizes the 5’ Cap of T3.0 RNA and scans down the T3.0 RNA. A small portion of ribosomes initiate translation at vSP-1 AUG and terminate translation at its stop codon. The majority of S40 ribosomes bypass the vSP-1 start codon and form translation initiation complex at vSP-2 AUG and initiate translation, representing a leaky scanning translation mechanism.

Discussion

A large portion of eukaryotic transcripts are noncoding RNAs and many of them contain short open reading frames (sORFs). Recent studies found that some of the sORFs encode biologically functional small peptides or microPeptides. A few well-studied microPeptides have indicated that these small peptides have important regulatory roles in many fundamental events including development [16,17), viral reactivation [2], muscle performance [3], metabolism [21], DNA repair [22] and immune responses [18]. Although it was known for many years that hormonal short peptides play important physiological roles, small peptide is a completely different category of bioactive peptides. Hormonal short peptides are synthesized as large polypeptides, then processed by cleaving them into short peptides and secreted to extracellular media functioning as hormones, while microPeptides are directly translated from sORFs of nocoding RNAs (obviously mis-annotated) and execute their modulation functions in cells. Interestingly, polycistronic translation has been observed in most of the cases of microPeptides translated from previously annotated noncoding RNAs characterized so far in eukaryotes [2,6,15–17]. It suggests that polycistronic translation is common for microPeptide synthesis in eukaryotic cells, representing a unique and non-canonical translation mechanism in eukaryotic cells. Elucidation of this mechanism is vital for understanding of the regulation of microPeptide synthesis in eukaryotic cells. The current study aims to elucidate such mechanism.

T3.0, as a polycistronic viral RNA, can be translated to generate several microPeptides [2,6]. In this study, we demonstrated that vSP-1 and vSP-2 are translated from T3.0 via a unique adaption of leaky scanning. The Kozak sequence around the AUG codon of vSP-1 regulates the vSP-2 translation. The weak Kozak context of vSP-1 allows most of 40S ribosomes that recognize the 5’cap structure of T3.0 RNA bypass the vSP-1 AUG codon and assemble S80 translation initiation complex at the vSP-2 AUG codon (Fig. 4). This observation is consistent with a leaky scanning mechanism that has been reported to be employed by viruses to translate their polycistronic mRNAs [23–25]. The length of intercistroic space between vSP-1 and VSP-2 is inversely propositional to the efficiency of vSP-2 translation, further confirming the leaky scanning mechanism and ruling out the ribosome re-entry or re-initiation model. This is in contrast with the bicistronic KSHV mRNA, where an IRES is present within the coding region of ORF72 allowing ORF71 to translate in a cap-independent manner [26].

Most polycistronic RNAs encode more than two sORFs. For instance, T3.0 is known to carry four translatable sORFs [6]. Further research is warranted to understand the mechanisms that govern the translation of the downstream sORFs.

Highlights.

• Polycistronic translation is frequently observed for synthesis of microPeptides in eukaryotes.

• KSHV-encoded T3.0 transcript contains a number of sORFs and some are translatable.

• VSP-1 and vSP-2 microPeptides are translated from T3.0 through a leaky scanning mechanism.