Resolution of polycistronic RNA by SL2<i>trans</i>-splicing is a widely conserved nematode trait

Wenzel, Marius; Johnston, Christopher; Müller, Berndt; Pettitt, Jonathan; Connolly, Bernadette

doi:10.1261/rna.076414.120

Cited by 3 publications

(17 citation statements)

References 64 publications

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“…The parasite T. spiralis possesses a diverse and unusual set of 15 SLs that are encoded by up to 48 genes [31] and are spliced to about 30 % of all 16,380 genes [20]. Three out of these 15 SLs ( Tsp -SL2, Tsp -SL10 and Tsp -SL12) are SL2-type SLs specialised for resolving downstream genes in operons [20]. We downloaded genome PRJNA12603.WBPS10 and three RNA-Seq libraries from bioproject PRJNA510020 [20].…”

Section: Resultsmentioning

confidence: 99%

“…Three out of these 15 SLs ( Tsp -SL2, Tsp -SL10 and Tsp -SL12) are SL2-type SLs specialised for resolving downstream genes in operons [20]. We downloaded genome PRJNA12603.WBPS10 and three RNA-Seq libraries from bioproject PRJNA510020 [20]. SLIDR was run with parameter ’ to accommodate for the smaller distance of the Sm binding motif to the splice donor site [31].…”

Section: Resultsmentioning

confidence: 99%

“…By default, no SL1-type reads are allowed at downstream genes (i.e., SL2:SL1 = infinity), but a more relaxed SL2:SL1 ratio cutoff can be provided. The optimal cutoff is species-specific and could be identified empirically from inspecting the distribution of SL2:SL1 read ratios or from observed read ratios at known operonic genes [20]. Each tract of SL2-biased operonic genes then receives an upstream operonic gene that shows SL1-type bias or absence of SL trans -splicing (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The same SL2-type specialisation of some SLs for resolving downstream genes in operons exists in many other nematodes [15–20], but is not seen in other eukaryotic groups. For example, the platyhelminth Schistosoma mansoni and the tunicates Ciona intestinalis and Oikopleura dioica each possess only a single SL, which is used to resolve polycistronic mRNAs but is also added to monocistronic transcripts [21–23].…”

Section: Introductionmentioning

confidence: 99%

“…An equally important barrier is the difficulty in quantifying SL trans -splicing events genome-wide and in establishing functional links between these events and operonic gene organisation. The 5’ ends of RNA-Seq reads contain, in principle, enough information to quantify SL trans -splicing events [29, 35], and genome-wide patterns of SL trans -splicing events have been exploited to predict novel operons from SL splicing ratios in the nematodes Pristionchus pacificus and Trichinella spiralis [19, 20]. The SL-QUANT pipeline automates quantification of SL trans -splicing events from RNA-Seq data for C. elegans and can be manually reconfigured to accept SL sequences and genomes from other organisms [36].…”

Section: Introductionmentioning

confidence: 99%

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SLIDR and SLOPPR: Flexible identification of spliced leadertrans-splicing and prediction of eukaryotic operons from RNA-Seq data

Wenzel

Mueller

Pettitt

2020

Preprint

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BackgroundSpliced leader (SL) trans-splicing replaces the 5’ ends of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from a different genomic location. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons have independently evolved multiple times throughout Eukarya, but our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes.ResultsHere we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5’ read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogation of sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5’ SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that 1) SLIDR correctly identifies known SLs and often discovers novel SL variants; 2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons.ConclusionsSLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya, and improve gene discovery and annotation for a wide-range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be derived from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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SLIDR and SLOPPR: Flexible identification of spliced leadertrans-splicing and prediction of eukaryotic operons from RNA-Seq data

Wenzel

Mueller

Pettitt

2020

Preprint

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A novel, essentialtrans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

Fasimoye

Spencer

Soto-Martin

et al. 2022

Nucleic Acids Research

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Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

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SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

2021

Self Cite

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Background Spliced leader (SL) trans-splicing replaces the 5′ end of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from elsewhere in the genome. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons may have independently evolved multiple times throughout Eukarya, yet our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes. Results Here we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5′ read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogating corresponding SL RNA genes for sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5′ SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that (1) SLIDR correctly detects expected SLs and often discovers novel SL variants; (2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons. Conclusions SLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya and improve gene discovery and annotation for a wide range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be retrieved from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

show abstract

Resolution of polycistronic RNA by SL2trans-splicing is a widely conserved nematode trait

Cited by 3 publications

References 64 publications

SLIDR and SLOPPR: Flexible identification of spliced leadertrans-splicing and prediction of eukaryotic operons from RNA-Seq data

SLIDR and SLOPPR: Flexible identification of spliced leadertrans-splicing and prediction of eukaryotic operons from RNA-Seq data

A novel, essentialtrans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

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