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Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

ABSTRACT: We recently reported that serine-arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65, and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

ORGANISM(S): Human adenovirus 2

PROVIDER: GSE188652 | GEO | 2022/07/05

REPOSITORIES: GEO

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Discovery of pre-mRNA structural scaffold as a contributor to mammalian splicing code [In Vivo]

Project description:The specific recognition of splice signals at or near the exon-intron junctions is not explained by their weak conservation across the mammalian transcriptome and postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of a pre-mRNA guiding early spliceosomal components to the splice signal sequences. We find that mutation in non-cognate splice signal sequences of a model pre-mRNA substrate could impede recruitment of early spliceosomal components due to disruption of global structure of the pre-mRNA. We also find distribution of pre-mRNA segments potentially interacting with early spliceosomal component U1 snRNP across the intron, spatial proximity of 5′ and 3′ splice sites within the pre-mRNA scaffold, and an interplay between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components could recognize a three-dimensional structural scaffold beyond the short splice signal sequences and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This work provides a conceptual base to extend our understanding of prevalence, distribution, and splicing regulatory potential of recognizable three-dimensional structural scaffolds across the mammalian transcriptome.

2021-06-23 | GSE173177 | GEO

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code [in vitro]

Project description:The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML – a model pre-mRNA substrate – guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5′ and 3′ splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome.

2021-06-23 | GSE173175 | GEO

Transcriptome changes in splicing inhibited cells

Project description:In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA–pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

2010-09-29 | GSE24179 | GEO

U1 snRNP complex with cleavage and polyadenylation factors controls mRNA transcription termination

Project description:Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3’-end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1–CPAFs), that binds intronic PASs and suppresses PCPA. U1–CPAFs are distinct from U1-spliceosomal complexes; they include CPA’s three main subunits, CFIm, CPSF, and CstF, lack essential splicing factors, and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1–CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1–CPAFs’ switch from repressive to activated states. Our findings outline U1 telescripting mechanism and demonstrate U1’s unique role as central-regulator of pre-mRNA processing and transcription.

2019-07-31 | GSE135140 | GEO

Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

Project description:Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (Nuclear RNAi defective-2) and CCDC174 (Coiled-Coil Domain-Containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and unspliced 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use non-canonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice and accurate pre-mRNA splicing.

2023-09-07 | PXD029392 | Pride

Suppression of prp28-1 splicing defect by prp8-tes allele

Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect. Splicing specific microarrays were used to assess suppression of the prp28-1 splicing defect by a prp8 allele (prp8-tes) that suppresses prp28-1 cold-sensitivity

2014-01-01 | E-GEOD-42754 | biostudies-arrayexpress

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

Project description:The recognition of 5’ splice site (5’ ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5’ ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5’ ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5’ ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

2024-01-03 | GSE252348 | GEO

A genome-wide analysis indicates that yeast pre-mRNA splicing is predominantly post-transcriptional

Project description:Recent ChIP experiments indicate that spliceosome assembly and splicing can occur cotranscriptionally in S. cerevisiae. However, only a few genes have been examined, and all have long second exons. To extend these studies, we analyzed intron-containing genes with different second exon lengths, by ChIP as well as by whole-genome tiling arrays (ChIP-CHIP). The data indicate that U1 snRNP recruitment is independent of exon length. Recursive splicing constructs, which uncouple U1 recruitment from transcription, suggest that cotranscriptional U1 recruitment contributes to optimal splicing efficiency. In contrast, U2 snRNP recruitment as well as cotranscriptional splicing is deficient on short second exon-genes. We estimate that approximately 90% of endogenous yeast splicing is post-transcriptional, consistent with an analysis of post-transcriptional snRNP-associated pre-mRNA. Keywords: ChIP-CHIP

2007-01-22 | GSE6417 | GEO

Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

2023-05-04 | GSE179744 | GEO

Frac-Seq Analysis of ZFC3H1 Knockdown U2OS Cells

Project description:We have found that depletion of the zinc finger protein ZFC3H1 inhibits the nuclear retention of these RNAs. ZFC3H1 forms a complex with MTR4 and PABPN1 (the PolyAdenylation EXosome Targeting complex (Meola et al 2016, Ogami et al 2017) and likely recruits the nuclear exosome for RNA degradation. Recently, we found that depletion of U1-70K, one protein component of the U1 snRNP spliceosomal complex, inhibits nuclear retention of 5’SS motif containing RNAs. The U1 snRNP complex recognizes and binds to the 5’SS motif and in vitro studies showed that U1-70K directly interact with a similar 5’SS motif through U1 snRNA binding (Gunderson et al 1999). Interestingly, we found that U1 snRNP is involved in targeting 5’SS motif containing mRNAs to nuclear speckles shortly following RNA transcription. In contrast, ZFC3H1 is involved in nuclear speckle localization post-transcriptionally and actively retain RNAs in these structures. Together, our results suggest a model where U1 snRNP interacts with the RNA via the 5’SS motif and targets mature mRNAs to nuclear speckles. Specifically, it suggests that misprocessed RNAs can be targeted to speckles (or other nucleoplasmic foci) and that these foci serve as a platform to recruit the nuclear exosome via ZFC3H1. Our results highlight a splicing independent role of U1 snRNP and how mRNA nuclear export function in nuclear surveillance to prevent the translation of aberrant proteins.

2022-03-31 | GSE176144 | GEO

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