Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

doi:10.1534/g3.116.027508

Cited by 18 publications

(25 citation statements)

References 86 publications

(98 reference statements)

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“…In contrast to smu1-4 and cwc16a-1, the two GFP-weak mutants reported here, prp8-10 and rtf2-4 showed generally reduced splicing efficiency of GFP premRNA, leading to increased accumulation of the unspliced primary transcript. The overall reduced splicing efficiency in these mutants is consistent with core spliceosomal roles of PRP8 (Pleiss et al 2007) and possibly RTF2 (Larson et al 2016), although a regulatory role through ubiquitination has also been suggested for RTF2 (Sasaki et al 2015).…”

Section: Mechanistic Aspects Of Smu1 and Cwc16a Activity In Splicingsupporting

confidence: 69%

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

Kanno

Lin

et al. 2017

RNA

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To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana. In wild-type plants, three major splice variants issue from the GFP gene but only one represents a translatable GFP mRNA. Compared to wild-type seedlings, which exhibit an intermediate level of GFP expression, mutants identified in the screen feature either a "GFP-weak" or "Hyper-GFP" phenotype depending on the ratio of the three splice variants. GFP-weak mutants, including previously identified prp8 and rtf2, contain a higher proportion of unspliced transcript or canonically spliced transcript, neither of which is translatable into GFP protein. In contrast, the coilindeficient hyper-gfp1 (hgf1) mutant displays a higher proportion of translatable GFP mRNA, which arises from enhanced splicing of a U2-type intron with noncanonical AT-AC splice sites. Here we report three new hgf mutants that are defective, respectively, in spliceosome-associated proteins SMU1, SmF, and CWC16, an Yju2/CCDC130-related protein that has not yet been described in plants. The smu1 and cwc16 mutants have substantially increased levels of translatable GFP transcript owing to preferential splicing of the U2-type AT-AC intron, suggesting that SMU1 and CWC16 influence splice site selection in GFP pre-mRNA. Genome-wide analyses of splicing in smu1 and cwc16 mutants revealed a number of introns that were variably spliced from endogenous pre-mRNAs. These results indicate that SMU1 and CWC16, which are predicted to act directly prior to and during the first catalytic step of splicing, respectively, function more generally to modulate splicing patterns in plants.

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Section: Mechanistic Aspects Of Smu1 and Cwc16a Activity In Splicingsupporting

confidence: 69%

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

Kanno

Lin

et al. 2017

RNA

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“…The Drosophila homolog, Yantar, was identified in a genetic deletion screen of lethal recessive mutants for factors that regulate hemocyte development; loss of Yantar is pupal lethal but leads to overproduction of hemocytes in larvae (Sinenko et al 2004). In a reverse genetic screen for factors that affect splicing efficiency in Schizosaccharaomyces pombe, deletion of the fission yeast homolog SPCC162.01c led to both the accumulation of unspliced forms of some messages and increased splicing efficiency for others (Larson et al 2016). SNRNP27 ′ s high enrichment in arginine and lysine residues leaves a very limited number of peptides that can be observed in mass spectrometry proteomic experiments after tryptic digest.…”

Section: Introductionmentioning

confidence: 99%

SNRP-27, the C. elegans homolog of the tri-snRNP 27K protein, has a role in 5′ splice site positioning in the spliceosome

Zahler

Rogel

Glover

et al. 2018

RNA

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The tri-snRNP 27K protein is a component of the human U4/U6-U5 tri-snRNP and contains an N-terminal phosphorylated RS domain. In a forward genetic screen in , we previously identified a dominant mutation, M141T, in the highly-conserved C-terminal region of this protein. The mutant allele promotes changes in cryptic 5' splice site choice. To better understand the function of this poorly characterized splicing factor, we performed high-throughput mRNA sequencing analysis on worms containing this dominant mutation. Comparison of alternative splice site usage between the mutant and wild-type strains led to the identification of 26 native genes whose splicing changes in the presence of the mutation. The changes in splicing are specific to alternative 5' splice sites. Analysis of new alleles suggests that is an essential gene for worm viability. We performed a novel directed-mutation experiment in which we used the CRISPR-cas9 system to randomly generate mutations specifically at M141 of SNRP-27. We identified eight amino acid substitutions at this position that are viable, and three that are homozygous lethal. All viable substitutions at M141 led to varying degrees of changes in alternative 5' splicing of native targets. We hypothesize a role for this SR-related factor in maintaining the position of the 5' splice site as U1snRNA trades interactions at the 5' end of the intron with U6snRNA and PRP8 as the catalytic site is assembled.

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“…Recently, we published the results of a quantitative screen for mutant strains which display splicing defects (Larson et al 2016). Using a sequencing-based approach, we were able to precisely and systematically measure the splicing efficiency of two endogenous transcripts in the background of ~3000 strains, each containing a deletion of a single, non-essential S. pombe gene.…”

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confidence: 99%

The power of fission: yeast as a tool for understanding complex splicing

Fair

Pleiss

2016

Curr Genet

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Pre-mRNA splicing is an essential component of eukaryotic gene expression. Many metazoans, including humans, regulate alternative splicing patterns to generate expansions of their proteome from a limited number of genes. Importantly, a considerable fraction of human disease causing mutations manifest themselves through altering the sequences that shape the splicing patterns of genes. Thus, understanding the mechanistic bases of this complex pathway will be an essential component of combating these diseases. Dating almost to the initial discovery of splicing, researchers have taken advantage of the genetic tractability of budding yeast to identify the components and decipher the mechanisms of splicing. However, budding yeast lacks the complex splicing machinery and alternative splicing patterns most relevant to humans. More recently, many researchers have turned their efforts to study the fission yeast, Schizosaccharomyces pombe, which has retained many features of complex splicing, including degenerate splice site sequences, the usage of exonic splicing enhancers, and SR proteins. Here, we review recent work using fission yeast genetics to examine pre-mRNA splicing, highlighting its promise for modeling the complex splicing seen in higher eukaryotes.

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Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

Cited by 18 publications

References 86 publications

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

SNRP-27, the C. elegans homolog of the tri-snRNP 27K protein, has a role in 5′ splice site positioning in the spliceosome

The power of fission: yeast as a tool for understanding complex splicing

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