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. Author manuscript; available in PMC: 2022 Jun 20.
Published in final edited form as: Mol Cell. 2022 Apr 28;82(11):1981–1991. doi: 10.1016/j.molcel.2022.04.008

It’s a DoG-eat-DoG world—altered transcriptional mechanisms drive downstream-of-gene (DoG) transcript production

Marc Morgan 1, Ramin Shiekhattar 2, Ali Shilatifard 1, Shannon M Lauberth 1,*
PMCID: PMC9208299  NIHMSID: NIHMS1814539  PMID: 35487209

SUMMARY

The past decade has revolutionized our understanding of regulatory noncoding RNAs (ncRNAs). Among the most recently identified ncRNAs are downstream-of-gene (DoG)-containing transcripts that are produced by widespread transcriptional readthrough. The discovery of DoGs has set the stage for future studies to address many unanswered questions regarding the mechanisms that promote readthrough transcription, RNA processing, and the cellular functions of the unique transcripts. In this review, we summarize current findings regarding the biogenesis, function, and mechanisms regulating this exciting new class of RNA molecules.

INTRODUCTION

Fifty-two years ago, our understanding of transcriptional regulation was transformed by the discovery of three nuclear RNA polymerases (Roeder, 2019). Since this monumental breakthrough, numerous studies have advanced our understanding of the highly regulated steps of the RNA polymerase II (RNAPII) transcription cycle, including initiation, elongation, and termination. The initiation step involves core promoter recognition and assembly of the preinitiation complex (PIC) that controls DNA opening and the start of RNAPII-mediated transcription (Buratowski, 1994; Conaway and Conaway, 1993; Jiang et al., 1996; Nikolov and Burley, 1997; Orphanides et al., 1996; Petrenko and Struhl, 2021; Roeder, 1996; Schier and Taatjes, 2020). Following initiation, RNAPII undergoes promoter-proximal pausing, a process that is regulated by several factors that stabilize paused RNAPII and control its release into productive elongation (Chen et al., 2018; Core and Adelman, 2019; Jonkers et al., 2014; Laitem et al., 2015; Mayer et al., 2015; Rahl et al., 2010; Wissink et al., 2019). Finally, upon reaching the 3′ end of genes, elongating RNAPII transitions into transcription termination, a regulatory step that involves RNA cleavage, polyadenylation, and RNAPII release (Core et al., 2008; Davidson et al., 2014; Fong et al., 2015; Hsin and Manley, 2012; Mandel et al., 2006; Meinhart and Cramer, 2004; Miki et al., 2017; Proudfoot, 2011, 2016; Tian and Manley, 2013; Zaborowska et al., 2016; Zhang et al., 2015). Numerous reviews have detailed the intricate regulatory steps of the transcription cycle (Core and Adelman, 2019; Dollinger and Gilmour, 2021; Porrua and Libri, 2015; Proudfoot, 2016; Schier and Taatjes, 2020). In addition to its role in the production of protein-coding mRNAs, RNAPII is also responsible for the production of noncoding RNAs (ncRNAs) that execute a diverse array of cellular functions (Cech and Steitz, 2014).

DoG RNAs REPRESENT A UNIQUE CLASS OF RNAPII-TRANSCRIBED ncRNAs

Downstream-of-gene (DoG)-containing RNAs are a unique class of ncRNA that possesses three defining features (Figure 1) (Rosa-Mercado and Steitz, 2022; Vilborg and Steitz, 2017). First, DoG RNAs initiate at the promoter of a protein-coding host gene. Second, DoG transcripts extend as long continuous transcripts for at least 5 kb beyond the 3′ terminal polyadenylation signals (PASs) of their host gene. Third, the DoG RNAs that have been studied to date are retained in the nucleus and are likely to remain associated with chromatin (Vilborg et al., 2015; Vilborg et al., 2017), although it is unknown if this is a universal feature. These properties set DoGs apart from many other ncRNAs, which do not originate from protein-coding genes and may have well-defined termination sites. Moreover, the production of DoG RNAs is triggered by various cellular stress responses and involves a disruption of RNAPII transcription termination and RNA polyadenylation (Grosso et al., 2015; Heinz et al., 2018; Nemeroff et al., 1998; Rosa-Mercado et al., 2021; Rutkowski et al., 2015; Shalgi et al., 2014; Vilborg et al., 2015; Vilborg and Steitz, 2017).

Figure 1. Molecular features that define readthrough transcripts (DoGs).

Figure 1.

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Schematic diagram to depict DoG biogenesis that occurs in response to various stress stimuli and in cancer versus normal tissue (Cardiello et al., 2018; Cugusi et al., 2022; Heinz et al., 2018; Nemeroff et al., 1998; Rosa-Mercado et al., 2021; Rutkowski et al., 2015; Shalgi et al., 2014; Vilborg et al., 2015; Vilborg et al., 2017; Vilborg and Steitz, 2017). DoGs are unidirectional, continuous transcripts that initiate from the promoter of the DoG-producing gene and extend at least >5 kb beyond the mRNA 3′ end processing polyadenylation (Poly-A) signals (Vilborg et al., 2015).

Pervasive transcription readthrough gives rise to the long DoG transcripts that are believed to account for 15%–30% of intergenic transcription (Rosa-Mercado and Steitz, 2022; Vilborg and Steitz, 2017). Unlike long intergenic ncRNAs (lincRNAs) that are autonomously transcribed, DoGs are transcripts generated from the upstream DoG-producing gene, which is capable of producing both spliced mRNA and RNA that is continuous with the long noncoding DoG transcript (Figure 1). This is best supported by a previous study (Vilborg et al., 2015), which revealed that DoG-producing genes contain only one transcription start site (TSS), as determined by CapSeq (Illingworth et al., 2010). Moreover, antisense oligonucleotide (ASO)-mediated knockdown of mRNA, synthesized from the DoG-producing host gene, results in a corresponding change in DoG levels, which is consistent with DoG production being dependent on continued transcription from the host gene promoter (Vilborg et al., 2015). Also, whereas lincRNAs are expressed at levels that are positively correlated with the expression levels of nearby genes (Andersson et al., 2014; Cheng et al., 2015; Kaikkonen et al., 2013; Lai et al., 2015; Rahnamoun et al., 2018; Sanyal et al., 2012), DoGs are produced regardless of the transcriptional levels of their host gene (Rosa-Mercado et al., 2021). For example, DoGs were found to be produced from DoG-producing genes that are activated, repressed, or not changing in response to hyperosmotic stress (Rosa-Mercado et al., 2021). These findings suggest that DoG versus DoG host gene regulatory mechanisms are uncoupled.

ESTABLISHED MECHANISMS REGULATING DoG RNA EXPRESSION

Although a number of cellular processes and factors have been linked to DoG biogenesis, the full breadth of mechanisms underlying transcriptional readthrough and various DoG properties remains to be fully elucidated. Strong evidence linking cellular stress responses to the expression of DoGs suggests a rapid mechanism underlying the switch producing spliced and polyadenylated protein-coding transcripts to long ncRNAs. DoGs are induced in response to various stress stimuli including osmotic stress (Rosa-Mercado et al., 2021; Vilborg et al., 2015), heat shock (Cardiello et al., 2018; Cugusi et al., 2022; Shalgi et al., 2014; Vilborg et al., 2017), influenza A virus (IAV) (Heinz et al., 2018; Nemeroff et al., 1998), and herpes simplex virus (HSV) infection (Rutkowski et al., 2015). Precisely how each of these diverse stress signals triggers DoG RNA expression is not fully understood; however, several involve alterations in RNAPII termination and RNA processing factors.

DoG expression is driven by RNAPII termination defects

Termination of gene transcription remains the least well-understood regulatory step of the transcription cycle (Proudfoot, 2016). Yet, defects in termination are becoming more readily identified and quantified by high-throughput nascent RNA sequencing methods that reveal “readthrough transcription” that maps downstream of the canonical polyadenylation sites to the 3′ gene boundary of protein-coding genes. The majority of studies to date suggest that DoG expression is tightly linked to termination defects.

Transcription termination is linked to mRNA 3′ end processing by polyadenylation and cleavage factors (Bauer et al., 2018; Nemeroff et al., 1998; Wang et al., 2020). Specifically, the PAS within the nascent transcript is recognized by the cleavage and PAS specificity factor (CPSF) that contains CPSF73, an endonuclease that cleaves the nascent RNA (Chan et al., 2014). Consistent with DoG production resulting from reduced transcription termination is the finding that the loss of CPSF73 results in partial induction of DoGs (Vilborg et al., 2015). The depletion of additional polyadenylation/termination factors, including 5′–3′ exoribonuclease, Xrn2, has also been shown to result in an enrichment of DoG production (Eaton et al., 2018). In addition, DoG-producing genes exhibit a reduced frequency of strong PASs compared with non-DoG-producing genes, which leads to less efficient termination in these regions and the ability of RNAPII to remain engaged in processive elongation past the normal termination sites (Rosa-Mercado et al., 2021). Similarly, nuclear poly(A)-binding protein, Nab2, which is a regulator of co-transcriptional splicing, has also been implicated in 3′ end cleavage of nascent RNA (Alpert et al., 2020). Consistent with this role, the loss of Nab2 was found to coincide with aberrant cleavage and termination, which in turn resulted in DoG production (Alpert et al., 2020).

In addition to canonical cleavage and polyadenylation factors, the Integrator complex is involved in regulating DoG expression in the context of cellular stress (Figure 2). The Integrator complex is a large macromolecular assembly that possesses a ribonuclease catalytic activity that is involved in an array of RNA-based processes including RNA 3′ end processing, RNAPII pause-release, and termination (Kirstein et al., 2021). Integrator directly interacts with RNAPII by binding to phosphorylation sites in its C-terminal domain (CTD) (Baillat et al., 2005; Egloff et al., 2010). Profiling of the RNAPII protein interactome under the condition of osmotic shock revealed that this cellular stress triggers the dissociation of the Integrator complex from RNAPII (Rosa-Mercado et al., 2021). Importantly, several groups have demonstrated that disrupting the function of Integrator subunit INTS11 is sufficient to induce DoG expression (Dasilva et al., 2021; Rosa-Mercado et al., 2021). Thus, Integrator function appears to be a key player in the suppression of DoG expression under normal conditions, and its function is perturbed during osmotic stress, leading to DoG RNA production.

Figure 2. Mechanisms driving DoG expression.

Figure 2.

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(A) Integrator is involved in the suppression of DoG expression. Under normal conditions, the Integrator complex associates with RNAPII CTD through interaction with the Serine 2 and Serine 7 double phosphorylation (S2P/S7P) mark (Egloff et al., 2010). During hyperosmotic stress, Integrator dissociates from RNAPII, leading to defective termination and the production of termination readthrough DoG RNAs (Rosa-Mercado et al., 2021). The phosphorylation status of the RNAPII CTD at DoG-producing genes during osmotic stress has not been determined; however, alteration in CTD phosphorylation could contribute to this process. PAS, polyadenylation site.

(B) Degradation of BRD4 triggers DoG expression. P-TEFb can be recruited to target genes through interaction with bromodomain-containing protein 4 (BRD4), which is a reader of acetylated nucleosomes (Peterlin and Price, 2006). P-TEFb promotes transcriptional elongation by phosphorylation of the Serine 2 residue of the RNAPII CTD (Peterlin and Price, 2006; Yang et al., 2005). In addition to binding to P-TEFb, BRD4 interacts with cleavage and polyadenylation specificity factor (CPSF) (Arnold et al., 2021). When BRD4 is degraded by the small molecule dBET6, this results in dissociation of CPSF from chromatin and causes an accumulation of DoG RNAs (Arnold et al., 2021). In the absence of BRD4-P-TEFb complexes, it is likely that super elongation complex (SEC)-P-TEFb is responsible for driving transcription elongation of DoGs through RNAPII S2 phosphorylation (Luo et al., 2012).

DoG expression and human disease

In addition to activation of DoGs by cellular stress responses, DoGs have also been linked to human disease. Correlative evidence in clear cell renal cell carcinoma (ccRCC) suggests that inactivation of the histone H3 lysine 36 (H3K36) methyltransferase, SETD2, may promote DoG production (Grosso et al., 2015). SETD2 is highly mutated in renal cancer and its loss is associated with a dramatic decrease of the H3K36me3 histone modification (Cancer Genome Atlas Research, 2013; Dalgliesh et al., 2010; Duns et al., 2010; Grosso et al., 2015; Varela et al., 2011). H3K36me3 is typically associated with the bodies of actively transcribed genes, and several studies have demonstrated its involvement in RNA processing through the recruitment of RNA processing factors (Fahey and Davis, 2017; Kim et al., 2011; Kolasinska-Zwierz et al., 2009). Intriguingly, in ccRCC SETD2, loss is correlated with the production of DoG RNAs (Grosso et al., 2015). Recent studies in Drosophila have also linked the H3K36me3 modification to RNA polyadenylation, suggesting a potential mechanism for SETD2’s involvement in DoG expression (Meers et al., 2017). However, it will be important to examine DoG expression in isogenic SETD2 loss-of-function experiments to establish a direct link between SETD2, H3K36me3, and DoGs.

It has recently been demonstrated that treatment of cancer cells with the bromodomain-containing protein 4 (BRD4) degrader dBET6 can trigger the expression of DoGs (Arnold et al., 2021) (Figure 2). In recent years, inhibitors and degraders of BRD4 have emerged as a promising cancer therapy (Filippakopoulos et al., 2010; Shi and Vakoc, 2014; Winter et al., 2017). BRD4 is recruited to chromatin through an interaction between its bromodomains and acetylated histones (Filippakopoulos et al., 2010). BRD4 functions as an RNAPII elongation factor (EF) in complex with the RNAPII CTD kinase P-TEFb, and its inhibition results in a variety of transcriptional defects (Jang et al., 2005; Shi and Vakoc, 2014; Yang et al., 2005). BRD4 degradation through the proteolysis-targeting chimeric (PROTAC) molecule dBET6, or by genetic tagging of BRD4 with an inducible degron tag (dTag), triggers widespread transcriptional termination defects resulting in DoG expression (Arnold et al., 2021). Moreover, these effects are dependent on BRD4 protein degradation, as opposed to BRD4 inhibition, as treatment of cells with the BRD4 bromodomain inhibitor JQ1 does not result in DoG expression, implying that these effects require almost complete loss of BRD4 function. Mechanistically, BRD4 interacts with RNA 3′ end processing factors such as CPSF as well as RNAPII EFs including PAF, DRB sensitivity inducing factor (DSIF), and NELF, and upon BRD4 degradation, these proteins exhibit severely impaired binding to chromatin (Arnold et al., 2021). This study revealed an unexpected link between an RNAPII EF and the control of termination. It will be important for future studies to examine if DoGs play a functional role in the cellular response to BRD4 degradation.

MECHANISMS INVOLVED IN RNA BIOGENESIS WITH POTENTIAL LINKS TO DoG EXPRESSION

Termination defects are clearly central to the production of DoG RNAs. However, there are a number of other transcriptional regulatory pathways that may be relevant to DoGs and are worthy of further examination. It is widely accepted that mRNA processing through splicing, polyadenylation, and capping occurs co-transcriptionally and is tightly linked to RNAPII elongation (Bentley, 2014; Neugebauer, 2002; Proudfoot et al., 2002). As these processes are molecularly coupled, this raises the possibility that alterations in termination, elongation, or splicing could potentially result in DoG expression. Consistent with this possibility, in both yeast and mammalian cells, unspliced mRNAs are typically not cleaved at their 3′ ends and often present as readthrough transcripts (Alpert et al., 2020; Dye and Proudfoot, 1999; Herzel et al., 2018; Reimer et al., 2021). Future studies will be required to define the splicing status of DoGs, which currently remains unclear.

RNAPII CTD phosphorylation recruits Integrator

Alterations of RNAPII CTD phosphorylation may be involved in DoG production with respect to Integrator recruitment. Post-translational modifications (PTMs) of the RNAPII CTD coordinate RNAPII activity by facilitating its association with a number of different factors, including transcription elongation and termination factors (Hintermair et al., 2012; Hsin and Manley, 2012; Mayer et al., 2012; Zaborowska et al., 2016). The RNAPII CTD also recruits RNA processing factors that do not necessarily coordinate RNAPII transcriptional activity (Hsin and Manley, 2012; Zaborowska et al., 2016). The RNAPII CTD consists of 52 heptad repeats of the consensus sequence (YSPTSPS) and is phosphorylated at Y1, S2, T4, S5, and S7 (Hirose and Manley, 2000; Proudfoot et al., 2002; Shatkin and Manley, 2000). Phosphorylated S2/S5 residues serve as hallmarks for active transcription of protein-coding genes (Corden, 1990; Eick and Geyer, 2013; West and Corden, 1995). Specifically, S5P levels are enriched at the 5′ ends of coding regions, and as RNAPII elongates, S5P levels decrease while S2P levels increase (Corden, 1990; Eick and Geyer, 2013; West and Corden, 1995). RNAPII CTD Y1 phosphorylation is predominantly localized in the antisense orientation at promoter regions and is significantly enriched at active enhancers (Hsin et al., 2014). CTD S7 phosphorylation is associated with RNAPII-transcribed small nuclear RNAs (snRNAs) and is required for their expression (Egloff and Murphy, 2008; Egloff et al., 2007).

The Integrator complex was initially characterized through its ability to interact with the RNAPII CTD (Baillat et al., 2005). Considering that osmotic stress impairs the interaction between RNAPII and Integrator (Rosa-Mercado et al., 2021), it is likely that RNAPII CTD PTMs may play a role in this process. Integrator binds most efficiently to a S2P/S7P double modification, which is implemented by a combination of P-TEFb (S2P) and DNA-PK (S7P) kinase activity (Egloff et al., 2010). Considering that BRD4 degradation disrupts the function of a subset of P-TEFb-containing complexes, it will be important to investigate whether DoG expression is linked to defective Integrator-RNAPII interactions similarly to osmotic stress. Recent studies have also implicated RNAPII CTD Y1 phosphorylation as being involved in Integrator recruitment. Mutation of Y1 to phenylalanine (Y1F) in 39 of the 52 heptad repeats results in widespread transcription termination readthrough leading to DoG production (Shah et al., 2018). The Y1F RNAPII also displays impaired interactions with the Integrator complex, although a direct interaction between Integrator and Y1P has not been demonstrated (Shah et al., 2018). Another caveat of this work is that the Y1F mutant may not fully mimic the non-phosphorylated tyrosine residue and also will disrupt any function that the unmodified tyrosine residue plays. Therefore, a more definitive answer to the role of Y1 phosphorylation will require the inhibition of the Y1 kinase.

RNAPII elongation factors in DoG expression

DoG expression requires both a failure to terminate normally as well as continued RNAPII elongation far beyond the normal gene 3′ end. This implicates RNAPII EFs as potential players in DoG biogenesis. Indeed, a molecular shift that favors continued transcriptional elongation over termination would be expected to promote DoG-producing expression. A large number of EFs are dedicated to the control of various aspects of RNAPII elongation (Chen et al., 2018; Zhou et al., 2012). Whereas many EFs, such as the super elongation complex (SEC) and P-TEFb, function as positive EFs that predominantly stimulate the elongation process, others exhibit more dynamic roles in regulating elongation (Chen et al., 2018; Sims et al., 2004; Zhou et al., 2012). For instance, DSIF, composed of the SPT4 and SPT5 subunits, inhibits elongation until it is phosphorylated by the CDK9 kinase, which converts it to a positive EF (Chen et al., 2018; Zhou et al., 2012). In addition, more recent studies have revealed an additional role for SPT5 in preventing proteasomal degradation of the RNAPII large subunit, Rpb1 (Aoi et al., 2021; Hu et al., 2021). Similarly, the RNAPII-associated factors complex (PAFc) associates with elongating polymerase and was presumed to function largely as a positive EF, but it was recently demonstrated that depletion of PAFc results in pause-release from a subset of genes (Chen et al., 2015; Chen et al., 2017). Thus, although a large number of factors have been implicated in regulating RNAPII elongation through in vitro assays, the complex function and dynamic regulation of EFs in a cellular context is only beginning to become understood.

P-TEFb, in addition to its crucial role in promoting RNAPII elongation through phosphorylation of S2 of the RNAPII CTD, also phosphorylates a number of EFs, including SPT5 and NELFE (Egloff, 2021), to promote transcriptional elongation. P-TEFb also exists in several distinct complexes (Peterlin and Price, 2006). For example, if P-TEFb is bound to the 7SK snRNA and HEXIM1/2 RNA-binding proteins, then it is maintained in an inactive pool that is available for release and incorporation into other complexes (Peterlin and Price, 2006). P-TEFb can also complex with both SEC and BRD4 (Jonkers and Lis, 2015), although these complexes likely serve distinct functions, with SEC being recruited to rapidly induced stimulus-responsive genes and BRD4 targeting to chromatin through its histone lysine acetylation reader bromodomains (Jang et al., 2005; Yang et al., 2005). Since BRD4 and SEC represent the two major activating P-TEFb-containing complexes, the observation that degradation of BRD4 results in activation of DoG expression strongly suggests that SEC is responsible for driving elongation of DoG RNAs.

THE FUNCTIONAL ROLES OF READTHROUGH TRANSCRIPTION AND DoGs

Despite DoGs becoming increasingly identified as a hallmark of termination defects (Heinz et al., 2018; Nemeroff et al., 1998; Rosa-Mercado et al., 2021; Rutkowski et al., 2015; Shalgi et al., 2014; Vilborg et al., 2015), it remains to be resolved whether readthrough transcription, DoGs themselves, or both exhibit consequences on cellular function. To date, there are few studies demonstrating that readthrough transcription can exhibit severe consequences on gene expression (Greger and Proudfoot, 1998; Shearwin et al., 2005). One such example is when the resulting readthrough transcripts run into the promoter of a nearby gene or ncRNA and restrict its activity through transcriptional interference or lead to the production of RNA chimeras (Greger and Proudfoot, 1998; Shearwin et al., 2005). Chimeric transcripts spanning multiple genes with retained introns have been shown to result when there is a failure in splicing due to a disruption in Nab2, which is required to ensure proper 3′ end cleavage (Alpert et al., 2020; Herzel et al., 2018) (Figure 3). In addition to transcriptional interference, the act of readthrough transcription has been shown to contribute to host genome 3D organization following IAV infection (Heinz et al., 2018). Specifically, an investigation of chromatin changes following IAV infection revealed that non-structural (NS1) protein of IAV induces readthrough transcription of highly active genes that results in the displacement of cohesin from chromatin, elimination of chromatin loops, and decompacted chromatin in the readthrough regions (Heinz et al., 2018) (Figure 3). These NS1-dependent changes in chromatin may contribute to NS1-dependent IAV virulence. Also consistent with a role for NS1 in contributing to transcription termination defects is the finding that NS1 inhibits the CPSF complex and disrupts mRNA cleavage and polyadenylation (Noah et al., 2003). Further studies are required to determine the relative contributions of NS1-dependent regulation of mRNA processing, pervasive readthrough transcription, and chromatin structure, and its ability to subvert host antiviral responses and increase virulence.

Figure 3. The roles of readthrough transcription and DoGs.

Figure 3.

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Schematic representation of the impact that readthrough transcription and DoGs have on the production of RNA chimeras, 3D chromatin organization, and nuclear scaffold reinforcement. In support of readthrough transcription leading to chimeric transcripts are several studies revealing aberrant coupling of splicing and 3′ end cleavage that leads to readthrough transcription and nascent transcripts with retained introns (Herzel et al., 2018). It has also been demonstrated that readthrough transcription leading to RNA chimeras originate following the depletion of nuclear export and splicing regulator, Nab2 (Alpert et al., 2020). RNAPII elongation has also been linked to chromatin structure in mammalian cells. Specifically, this was demonstrated by a study revealing influenza A virus (IAV)/NS1-dependent inhibition of transcription termination in which RNAPII elongates past termination sites leading to a loss of chromatin loops and local chromatin decompaction (Heinz et al., 2018). Emerging evidence also suggests that the DoGs themselves may function by reinforcing the nuclear scaffold during stress responses. This was supported by a study revealing that preventing DoG induction after osmotic stress, which leads to nuclear shrinkage as water is forced from the cell (Finan and Guilak, 2010), results in a more severe level of nuclear shrinkage and chromatin collapse (Vilborg et al., 2015). Together, these recent findings reveal the importance of further dissecting the relationship between readthrough transcription and DoGs to understand key elements of their functional mechanisms.

The identification that readthrough transcription through DoG-producing regions is a regulated process suggests that DoGs are functional (Alpert et al., 2020; Arnold et al., 2021; Cardiello et al., 2018; Dasilva et al., 2021; Grosso et al., 2015; Hennig et al., 2018; Melnick et al., 2019; Rosa-Mercado et al., 2021; Sharma et al., 2014). To our knowledge, however, there are no studies to date that have directly manipulated the DoG itself to discern its direct (causal) functions. Thus, it largely remains a mystery as to whether DoGs are functional molecules in the cell. DoGs have been shown to be found in the chromatin-bound fraction where they appear to colocalize with their upstream DoG-producing mRNA (Vilborg et al., 2015). This finding, together with the large size of DoGs, suggests that they may be involved in nuclear scaffold reinforcement. Consistent with this possibility is the finding that disrupting DoG induction by IP3R inhibition aggravates osmotic-stress-associated chromatin collapse (Vilborg et al., 2015) (Figure 3). Additional investigation is needed to determine whether DoGs affect gene expression in addition to maintaining nuclear integrity and stabilizing genomic regions that support DoG production.

FUTURE PERSPECTIVES

Readthrough transcription is an emerging phenotype, and DoG discovery opens the door for an exciting time defined by cataloging additional DoGs, identifying various DoG properties, and determining the particular genes that are subject to transcription readthrough in various cellular conditions and organisms of interest. In addition, it will be important for the field to provide key insights into unrecognized mechanisms underlying transcriptional readthrough and determine whether DoGs exhibit functions that impact cellular processes.

The process of cataloging additional DoGs across tissue types, developmental stages, and disease states, and distinguishing them from other transcripts, will be an important but also challenging effort because of the read-in transcription of DoGs into neighboring genes (Cardiello et al., 2018; Dasilva et al., 2021; Hennig et al., 2018; Rosa-Mercado et al., 2021; Roth et al., 2020; Rutkowski et al., 2015). Proper assignment of DoGs in different conditions and cell types requires the ability to identify DoGs that do not overlap with neighboring regions that are transcriptionally active on either DNA strand. As demonstrated in Table 1, we highlight a few of the packages that have been developed to streamline the process of DoG identification and quantification (Melnick et al., 2019; Roth et al., 2020; Wiesel et al., 2018). The recent development of long-read sequencing technologies will allow for the sequencing of full-length DoG transcripts, resolving issues such as the splicing status of DoGs and determining whether these transcripts are enriched or depleted for RNA modifications (Logsdon et al., 2020; Lorenz et al., 2020; Yue et al., 2015). There is also an imminent need to develop tools to experimentally manipulate and examine the direct functions of the plethora of ncRNAs that include DoGs. With an important consideration of any of these methods comes the challenge of discerning the functions of specific DoGs, while ensuring to discount any potential bleed through of another neighboring transcript. In Table 2, we highlight some of the commonly used practices in the field for studying RNA function that could be applied to probing the potential functional significance of DoGs (Abudayyeh et al., 2017; Arun et al., 2016; Bensaude, 2011; Daneshvar et al., 2020; Lai et al., 2020; Lee and Mendell, 2020; Leppek and Stoecklin, 2014; McHugh and Guttman, 2018; Rahnamoun et al., 2018; Vilborg et al., 2015). Taken together, this information will enable researchers to explore the readthrough phenomenon and advance our understanding of the widespread transcriptional readthrough that is arising in response to various stress conditions in mammalian cells.

Table 1.

Tools for identifying readthrough transcription in a variety of systems and contexts

Tool Tool Description References
DoGFinder Python based software package that identifies DoGs and quantifies their expression levels from any RNA-seq dataset. DoGFinder identifies DoGs by requiring a minimal coverage over a minimal initial length of continuous read density downstream of the 3’ end of every gene locus. Wiesel et al., 2018
Automatic Readthrough Transcription Detection (ARTDeco) Python based software that provides the framework for quantification and characterization of readthrough transcription from NGS data. ARTDeco quantifies the degree of readthrough transcription using read-in and readthrough levels and the detection of DoG transcripts.
ARTDeco varies from other programs by considering the discovered transcripts that extend into annotated genes to avoid the potential of arbitrary truncation.		 Roth et al., 2020
Dogcatcher Python based software that identifies DoG locations, genes that overlap with DoGs on the same or opposite strand, and an optional pipeline to provide differential expression of DoGs. Melnick et al., 2019
Long-read RNA-sequencing platforms Entire nascent transcripts are sequenced from the 5’ end (defined by the transcription start site [TSS]) to the 3’ end (defined as the position of RNA Pol II at the time of isolation). Logsdon et al., 2020
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Table 2.

Tools for exploring functional significance of DoGs

Tool Tool description References
Inhibiting eukaryotic transcription using widely used compounds Actinomycin D targets DNA and inhibits RNAPll elongation. α-amanitin targets RNAPll to inhibit RNA synthesis, and CDK9 inhibitors such as DRB and flavopiridol inhibit RNAPll elongation and rRNA processing. Yue et al., 2015
RNAi-mediated knockdown or incorporation of antisense oligonucleotides (ASOs) Loss-of-function studies have been instrumental in discerning ncRNA functions. ASOs revealed that knocking down mRNA of the host DoG-producing genes results in reduced levels of the corresponding DoGs. Thus, ASOs could be particularly useful with the targeting of antisense oligos to facilitate disruptions in termination at different regions of the DoG-producing host gene since ASOs are known to result in premature transcription termination. Vilborg et al., 2015
Rahnamoun et al., 2018
Bensaude, 2011
Arun et al., 2016
Lai et al., 2020
Cas13 Rnase The Cas9 system has been deployed in mammalian cells to target the degradation of specific RNA molecules, including lncRNAs. The potential advantages over oligonucleotide-based DoG depletion systems is in terms of limiting off-target effects and may be applicable to a wide-array of biological systems because it can be genetically encoded. Moreover, catalytically dead Cas13 is able to bind to its target RNAs without degrading them and thus would provide a useful tool for visualizing DoG transcripts and could potentially be used to disrupt DoG interactions with RNA binding proteins. Abudayyeh et al., 2017
RNA antisense purification (RAP) and streptavidin aptamers for purification of ribonucleoprotein complexes Proteome-wide screens for identifying the interactome of DoGs could be employed. This approach would take advantage of established methodologies that have been coupled with mass spectrometry (MS) to identify proteins that interact with lncRNAs in cells, including RNA antisense purification (RAP and streptavidin aptamers for purification of ribonucleoprotein complexes. Daneshvar et al., 2020
Leppek and Stoecklin, 2014 McHugh and Guttman, 2018
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ACKNOWLEDGMENTS

We thank Pedro Antonio Avila Lopez and Brianna Monroe for contributing to figure preparation for this review article. We apologize for any oversights or omissions. Research in the Lauberth lab is supported by a grant from the NIH/National Institute of General Medical Sciences (R35 GM128900) to S.M.L.; research in the Shilatifard laboratory is supported by NIH (R35CA197569 and U54CA231638) to A.S.; and research in the Shiekhattar laboratory is supported by funding from the University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center and grants R01 GM078455, R01 GM105754, P30 CA240139, and DP1 CA228041 from the National Institute of Health to R.S.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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