FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns

Ebersberger, Stefanie; Hipp, Clara; Mulorz, Miriam M.; Buchbender, Andreas; Hubrich, Dalmira; Kang, Hyun-Seo; Martínez-Lumbreras, Santiago; Kristofori, Panajot; Sutandy, F.X. Reymond; Llacsahuanga Allcca, Lidia; Schönfeld, Jonas; Bakisoglu, Cem; Busch, Anke; Hänel, Heike; Tretow, Kerstin; Welzel, Mareen; Di Liddo, Antonella; Möckel, Martin M.; Zarnack, Kathi; Ebersberger, Ingo; Legewie, Stefan; Luck, Katja; Sattler, Michael; König, Julian

doi:10.1016/j.molcel.2023.07.002

Cited by 19 publications

(8 citation statements)

References 139 publications

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“…Such regulation could add layers to gene expression programs: in addition to promoter-based and translational gene expression regulation, distinct subsets of splicing factors could influence the gene expression profile in different cell types as well. Indeed, some splicing proteins have been characterized to preferentially impact longer introns 64 . Such differential regulation could become an essential aspect of a cellular and/or developmental program, leading to the ‘functionalization’ of gigantic introns.…”

Section: Discussionmentioning

confidence: 99%

Co-transcriptional splicing facilitates transcription of gigantic genes

Fingerhut,

Lannes,

Whitfield

et al. 2024

Preprint

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Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of cotranscriptional splicing.

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Section: Discussionmentioning

confidence: 99%

Co-transcriptional splicing facilitates transcription of gigantic genes

Fingerhut,

Lannes,

Whitfield

et al. 2024

Preprint

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“…The resulting PRPF40A N-terminal region was cloned into a GFP-expressing pcDNA5 vector (see pcDNA5 GFP vector in ref. 28 ) using Q5 Site-Directed Mutagenesis Kit (NEB). Nter-MUT, containing the PPVP/PALPP motifs changed to AAAA/AAAAA, was obtained by mutating the N-ter-WW12-containing plasmid with Q5 site-directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

“…There are several interactions during the early stages of spliceosome assembly, which bridge the 5′ and 3′SS, both across the intervening intron and exon. For example, the stem loop 4 of the U1 snRNA (5′SS) is recognized by the UBL-like domain of SF3A1 (U2 snRNP component in 3′SS) 21 , 22 ; the UAP56 (RNA helicase) binds to the U1 snRNA stem loop 3 enhancing complex A formation 23 ; the SR proteins are thought to mediate U1 and U2 snRNPs bridging through their serine-arginine motifs 24 – 27 ; and recently, FUBP1 was identified as a core splicing factor that binds upstream of the BPS but also mediates interactions with the U1 snRNP 28 . The splicing factor PRPF40A was proposed to mediate a bridging connection already in complex E (prior binding of U2 snRNP) by the simultaneous recognition of U1 snRNP at the 5′ SS and SF1 at the BPS 29 , 30 .…”

Section: Introductionmentioning

confidence: 99%

Intramolecular autoinhibition regulates the selectivity of PRPF40A tandem WW domains for proline-rich motifs

Martínez-Lumbreras,

Träger,

Mulorz

et al. 2024

Nat Commun

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PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.

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“…FUBP1 is traditionally recognized for its role in Myc transcription regulation [ 28 ]. However, a recent report has shown that FUBP1 also functions as a general splicing factor, strengthening its potential as a NEAT1 interacting protein [ 29 ]. Similarly, Paraspeckles were initially identified in a search for nucleolar proteins [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

Multiple Oligo assisted RNA Pulldown via Hybridization followed by Mass Spectrometry (MORPH-MS) for exploring the RNA-Protein interactions

Pant,

Kumarswamy

2023

RNA Biology

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Understanding RNA-protein interactions is crucial for deciphering the cellular functions and molecular mechanisms of regulatory RNAs. Consequently, there is a constant need to develop innovative and cost-effective methods to uncover such interactions. We developed a simple and cost-effective technique called Multiple Oligo assisted RNA Pulldown via Hybridization (MORPH) to identify proteins interacting with a specific RNA. MORPH employs a tiling array of antisense oligos (ASOs) to efficiently capture the RNA of interest along with proteins associated with it. Unlike existing techniques that rely on multiple individually biotinylated oligos spanning the entire RNA length, MORPH stands out by utilizing a single biotinylated oligo to capture all the ASOs. To evaluate MORPH’s efficacy, we applied this technique combined with mass spectrometry to identify proteins interacting with lncRNA NEAT1, which has previously been studied using various methods. Our results demonstrate that despite being a simple and inexpensive procedure, MORPH performs on par with existing methods. Abbreviations : ASO, Antisense oligo; lncRNA, long non-coding RNA; MORPH, Multiple Oligo assisted RNA Pulldown via Hybridization

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FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns

Cited by 19 publications

References 139 publications

Co-transcriptional splicing facilitates transcription of gigantic genes

Co-transcriptional splicing facilitates transcription of gigantic genes

Intramolecular autoinhibition regulates the selectivity of PRPF40A tandem WW domains for proline-rich motifs

Multiple Oligo assisted RNA Pulldown via Hybridization followed by Mass Spectrometry (MORPH-MS) for exploring the RNA-Protein interactions

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