Lives that introns lead after splicing

Hesselberth, Jay R.

doi:10.1002/wrna.1187

Cited by 65 publications

(55 citation statements)

References 112 publications

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“…Thus, the lariat intron product offers a faithful, efficient, and direct alternative to mRNA for studying splicing genome-wide. Importantly, capturing the full landscape of introns, or the intronome, can provide insight into the production of not only mRNAs but also intron-encoded ncRNAs, such as small nucleolar RNAs, microRNAs, and some intronic circRNAs (Zhang et al 2013;Hesselberth 2014;Talhouarne and Gall 2014).…”

Section: Introductionmentioning

confidence: 99%

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Qin

Huang

Wlodaver

et al. 2015

RNA

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Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimalespecially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5 ′ splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3 ′ termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3 ′ to 5 ′ RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

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Section: Introductionmentioning

confidence: 99%

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Qin

Huang

Wlodaver

et al. 2015

RNA

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“…Despite the activity of the debranching pathway, a number of lariat-intermediates and lariat-introns are stable in cells (Hesselberth 2013). Furthermore, turnover efficiency among branched RNA products is variable.…”

Section: Introductionmentioning

confidence: 99%

A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Garrey

Katolik

Prekeris

et al. 2014

RNA

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Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudometallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5 ′ -exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.

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“…It has long been thought that the splicing process is very fast and spliced intronic RNA is rapidly degraded, but it is becoming clear that some introns have additional functions and escape the rapid degradation process. 36 Evidently, a part of the intronic RNA content in RNAomeSeq can be explained by the presence of precursor-mRNAs (pre-mRNAs). Secondly, some introns could be more stable than anticipated or, thirdly, functional non-coding RNAs could originate from intronic regions.…”

Section: Discussionmentioning

confidence: 99%

Deciphering the RNA landscape by RNAome sequencing

et al. 2015

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Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods.

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Lives that introns lead after splicing

Cited by 65 publications

References 112 publications

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Deciphering the RNA landscape by RNAome sequencing

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