Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5′ splice-site sequences due to weak interactions with SR proteins

Krchňáková, Zuzana; Thakur, Prasoon Kumar; Krausová, Michaela; Bieberstein, Nicole; Haberman, Nejc; Müller-McNicoll, Michaela; Staněk, David

doi:10.1093/nar/gky1147

Cited by 59 publications

(47 citation statements)

References 95 publications

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“…Pir2 and cryptic intron-based mechanisms repress targets throughout the genome and may be conserved in higher eukaryotes. Indeed, we note that ARS2 interacts with splicing factors in human cells, and lncRNAs such as XIST implicated in X-chromosome inactivation contain inefficiently spliced introns 47,48 . Considering that defects in lncRNAmediated gene regulation contribute to human diseases including cancer and that ARS2 is commonly mutated in various types of cancer, our findings may shed light on pathways contributing to the misexpression of genes, ultimately leading to the development of specific therapeutic strategies.…”

Section: Discussionmentioning

confidence: 91%

Conserved protein Pir2ARS2 mediates gene repression through cryptic introns in lncRNAs

et al. 2020

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Long non-coding RNAs (lncRNAs) are components of epigenetic control mechanisms that ensure appropriate and timely gene expression. The functions of lncRNAs are often mediated through associated gene regulatory activities, but how lncRNAs are distinguished from other RNAs and recruit effector complexes is unclear. Here, we utilize the fission yeast Schizosaccharomyces pombe to investigate how lncRNAs engage silencing activities to regulate gene expression in cis. We find that invasion of lncRNA transcription into the downstream gene body incorporates a cryptic intron required for repression of that gene. Our analyses show that lncRNAs containing cryptic introns are targeted by the conserved Pir2 ARS2 protein in association with splicing factors, which recruit RNA processing and chromatin-modifying activities involved in gene silencing. Pir2 and splicing machinery are broadly required for gene repression. Our finding that human ARS2 also interacts with splicing factors suggests a conserved mechanism mediates gene repression through cryptic introns within lncRNAs.

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Section: Discussionmentioning

confidence: 91%

Conserved protein Pir2ARS2 mediates gene repression through cryptic introns in lncRNAs

et al. 2020

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“…7c). Second, we inspected binding of RBPs to their own NMD transcripts using available eCLIP and iCLIP data [39][40][41] . In several cases, such as BCLAF1, TIAL1, SRSF5, SRSF6 and HNRNPL, a conserved PCE that generates Split-ORFs was massively bound by the encoded RBP (Fig.…”

Section: Rna Binding and Oligomerization Of Srsf7 Drive The Formationmentioning

confidence: 99%

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

Königs

Machado

Arnold

et al. 2020

Nat Struct Mol Biol

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Results SRSF7 overexpression induces auto-regulation.To investigate the mechanisms of SRSF7 homeostasis, we generated cell lines SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions. SRSF7 binding promotes splicing of NMD-sensitive and -resistant SRSF7 isoforms. To understand the mechanism of SRSF7 auto-regulation, we examined the binding of SRSF7 protein to SRSF7 transcripts using individual-nucleotide resolution ultraviolet (UV) crosslinking and immunoprecipitation (iCLIP). We used normalized significant crosslink events (X-links, false discovery rate (FDR) < 0.05) from SRSF3 and SRSF7 iCLIP datasets of P19 cell lines without OE 8 . Similar to SRSF3, which promotes the inclusion of the PCE in SRSF7 transcripts 17 , SRSF7 showed enriched crosslinks in an extended region encompassing the PCE, its flanking introns 3a and 3b, and exons 3, 4 and 5 ( Fig. 1d). Quantification revealed that SRSF7 binds ~50-fold more to SRSF7 transcripts than SRSF3 ( Supplementary Table 1), indicating that SRSF7 has an unusual preference for its own transcripts.RNA-seq followed by quantification of junction reads revealed that SRSF7 OE promotes inclusion of either the complete PCE (460 nucleotides (nt)) or a partial PCE (107 nt) in SRSF7 transcripts ( Fig. 1e). Additionally, both PCE-flanking introns (3a and 3b) and intron 5 displayed increased read coverage, indicating that they are partly retained upon SRSF7 OE. Semiquantitative reverse transcription PCR and sequencing confirmed that SRSF7 OE caused the appearance of transcripts containing the entire PCE (SRSF7-PCE, orange asterisk), the partial PCE (SRSF7-PCE 1/4 , yellow asterisk) or the PCE in combination with both flanking introns (SRSF7-I3a+b, red asterisk) or with only intron 3b (SRSF7-I3b, green asterisk; Fig. 1f and Extended Data Fig. 1b,c). Identical isoforms were detected for endogenous and SRSF7-GFP reporter transcripts, indicating that auto-regulation operates similarly on both.All these transcripts contain PTCs and should be s...

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“…Despite evidence that the functions of most elincRNAs is likely transcription dependent, a relatively large proportion of eli-ncRNAs is not only stably transcribed but also undergoes splicing (Marques et al, 2013;Hon et al, 2017;Krchnakova et al, 2019). Recently, splicing of Blustr, a lincRNA expressed in mouse embryonic stem cells (mESCs) whose transcriptional start site initiates from an active enhancer (Mouse ENCODE Consortium et al, 2012), was shown to be sufficient to modulate the expression of its cognate protein-coding gene target in cis (Engreitz et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Splicing of enhancer-associated lincRNAs contributes to enhancer activity

et al. 2020

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Transcription is common at active mammalian enhancers sometimes giving rise to stable enhancer-associated long intergenic noncoding RNAs (elincRNAs). Expression of elincRNA is associated with changes in neighboring gene product abundance and local chromosomal topology, suggesting that transcription at these loci contributes to gene expression regulation in cis. Despite the lack of evidence supporting sequence-dependent functions for most elincRNAs, splicing of these transcripts is unexpectedly common. Whether elincRNA splicing is a mere consequence of cognate enhancer activity or if it directly impacts enhancer function remains unresolved. Here, we investigate the association between elincRNA splicing and enhancer activity in mouse embryonic stem cells. We show that multi-exonic elincRNAs are enriched at conserved enhancers, and the efficient processing of elincRNAs is strongly associated with their cognate enhancer activity. This association is supported by their enrichment in enhancer-specific chromatin signatures; elevated binding of co-transcriptional regulators; increased local intra-chromosomal DNA contacts; and strengthened cis-regulation on target gene expression. Our results support the role of efficient RNA processing of enhancer-associated transcripts to cognate enhancer activity.

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Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5′ splice-site sequences due to weak interactions with SR proteins

Cited by 59 publications

References 95 publications

Conserved protein Pir2ARS2 mediates gene repression through cryptic introns in lncRNAs

Conserved protein Pir2ARS2 mediates gene repression through cryptic introns in lncRNAs

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

Splicing of enhancer-associated lincRNAs contributes to enhancer activity

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