Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Almada, Albert E.; Wu, Xuebing; Kriz, Andrea J.; Burge, Christopher B.; Sharp, Phillip A.

doi:10.1038/nature12349

Cited by 385 publications

(491 citation statements)

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“…ICP27's impact on polyadenylation from intronic PAS typically located within 1 kb of the 5′ splice site mirrors that recently observed when U1 snRNP's binding to the 5′ splice site was inhibited, also relieving its inhibition of CPSF binding to the downstream PAS (34)(35)(36). We hypothesize that ICP27 may thus interfere with U1 snRNP's binding to 5′ splice sites in the context of specific introns, through direct or indirect interaction with the C-rich sequences near the 5′ splice site (Fig.…”

Section: Splicing Inhibition Mediated By Icp27 and Cytosine-rich Sequmentioning

confidence: 60%

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner

Tang

Patel

Krause

2016

Proc. Natl. Acad. Sci. U.S.A.

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The herpes simplex virus (HSV) infected cell culture polypeptide 27 (ICP27) protein is essential for virus infection of cells. Recent studies suggested that ICP27 inhibits splicing in a gene-specific manner via an unknown mechanism. Here, RNA-sequencing revealed that ICP27 not only inhibits splicing of certain introns in <1% of cellular genes, but also can promote use of alternative 5′ splice sites. In addition, ICP27 induced expression of pre-mRNAs prematurely cleaved and polyadenylated from cryptic polyadenylation signals (PAS) located in intron 1 or 2 of ∼1% of cellular genes. These previously undescribed prematurely cleaved and polyadenylated pre-mRNAs, some of which contain novel ORFs, were typically intronless, <2 Kb in length, expressed early during viral infection, and efficiently exported to cytoplasm. Sequence analysis revealed that ICP27-targeted genes are GC-rich (as are HSV genes), contain cytosine-rich sequences near the 5′ splice site, and have suboptimal splice sites in the impacted intron, suggesting that a common mechanism is shared between ICP27-mediated alternative polyadenylation and splicing. Optimization of splice site sequences or mutation of nearby cytosines eliminated ICP27-mediated splicing inhibition, and introduction of C-rich sequences to an ICP27-insensitive splicing reporter conferred this phenotype, supporting the inference that specific gene sequences confer susceptibility to ICP27. Although HSV is the first virus and ICP27 is the first viral protein shown to activate cryptic PASs in introns, we suspect that other viruses and cellular genes also encode this function. (ICP27), an immediate early (IE) gene (among those first expressed after virus enters the cells) that is required for expression of some early and late viral genes as well as for virus growth, is highly conserved between HSV-1 and -2, two closely related neurotropic herpesviruses (1). ICP27 has a role in transcriptional regulation through association with the C-terminal domain of RNA polymerase II (2, 3), forms homodimers (4, 5), interacts with U1 small nuclear ribonucleoprotein (snRNP) through its C-terminal domain, and colocalizes with U1 and U2 snRNPs (6, 7). It also interacts with splicing factors such as SRSF3, SRSF1, SRSF7, and SRSF2 (8-11), and is involved in nuclear export of some viral transcripts (12, 13). The role of ICP27 in regulating pre-mRNA splicing remains controversial. Early studies indicated that, in an in vitro pre-mRNA splicing system, ICP27 may nonspecifically inhibit host pre-mRNA splicing, impairing spliceosome assembly as a result of interaction with SR protein kinase 1 (SRPK1) through ICP27's N-terminal RGG RNA-binding motif and/or interaction with spliceosome-association protein 145 (SAP145 or SF3B2) through ICP27's C-terminal domain (8, 11). A recent communication reported that HSV-1 does not inhibit cotranscriptional splicing and proposed that previous reports of ICP27-induced splicing inhibition were artifacts, due to misinterpretation of run-on transcription (14). Indeed, splicing of ...

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Section: Splicing Inhibition Mediated By Icp27 and Cytosine-rich Sequmentioning

confidence: 60%

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner

Tang

Patel

Krause

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…In some cases, not even the product but rather the act of transcription itself provides functionality: For instance, deep-sequencing has identified the existence of a class of divergent lncRNAs that are transcribed anti-sense to the promoters of coding genes and provide directionality of coding gene transcription (Almada et al, 2013). The resulting lncRNA products might simply represent byproducts of transcription and are typically rapidly degraded after synthesis (Clark et al, 2012).…”

Section: Eukaryotic Long Non-coding Rnas (Lncrnas)mentioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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“…Furthermore, a number of studies have demonstrated roles for U1 outside of pre-mRNA splicing. These include regulating the use of cleavage and polyadenylation sites (54) and conferring proper polarity to bidirectional transcription start sites (55). Whether any of these processes are affected in C. merolae remains to be determined.…”

Section: Dead Box Proteinsmentioning

confidence: 99%

Dramatically reduced spliceosome in Cyanidioschyzon merolae

Stark

Dunn²,

Dunn

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

143

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The human spliceosome is a large ribonucleoprotein complex that catalyzes pre-mRNA splicing. It consists of five snRNAs and more than 200 proteins. Because of this complexity, much work has focused on the Saccharomyces cerevisiae spliceosome, viewed as a highly simplified system with fewer than half as many splicing factors as humans. Nevertheless, it has been difficult to ascribe a mechanistic function to individual splicing factors or even to discern which are critical for catalyzing the splicing reaction. We have identified and characterized the splicing machinery from the red alga Cyanidioschyzon merolae, which has been reported to harbor only 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain expected conserved sequences and have the ability to adopt secondary structures and form intermolecular base-pairing interactions, as in other organisms. C. merolae has a highly reduced set of 43 identifiable core splicing proteins, compared with ∼90 in budding yeast and ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA candidate or any predicted U1-associated proteins, suggesting that splicing in C. merolae may occur without the U1 small nuclear ribonucleoprotein particle. In addition, based on mapping the identified proteins onto the known splicing cycle, we propose that there is far less compositional variability during splicing in C. merolae than in other organisms. The observed reduction in splicing factors is consistent with the elimination of spliceosomal components that play a peripheral or modulatory role in splicing, presumably retaining those with a more central role in organization and catalysis.pre-mRNA splicing | spliceosome core | U1 snRNP | genome reduction | splicing mechanism P re-mRNA splicing occurs by two transesterification reactions that are catalyzed by the spliceosome, a large macromolecular assembly of five snRNAs and more than 200 proteins in humans (1). These components are thought to assemble onto each new pre-mRNA transcript in an ordered fashion through the recognition and binding of three highly conserved sequences in the transcript: the 5′ splice site, the branch site, and the 3′ splice site (2, 3). Some of these interactions occur via direct RNA/RNA base pairing between the transcript and snRNAs; for example, both U1 and U6 snRNAs base pair to the 5′ splice site of the pre-mRNA transcript, and, similarly, U2 snRNA base pairs to the branch site (3).Given the complexity of the human spliceosome, it is of considerable interest to find a more tractable splicing system with fewer components to study the core processes of splicing (assembly, catalysis, and fidelity). The Saccharomyces cerevisiae (yeast) spliceosome has been proposed as a simplified model system, because it contains only about 100 proteins (4). Indeed, substantial progress in understanding the spliceosome has been made by studying yeast splicing (3,5). Nevertheless, the yeast spliceosome is still a highly complex system in which to investigate the role of individual proteins, let alone attempt ...

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Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Cited by 385 publications

References 35 publications

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner

Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner

Dual RNA-seq of pathogen and host

Dramatically reduced spliceosome in Cyanidioschyzon merolae

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