An <i>in vitro</i> reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands

Campagne, Sébastien; Vries, Tebbe de; Malard, Florian; Afanasyev, Pavel; Dorn, Georg; Dedic, Emil; Kohlbrecher, J.; Boehringer, Daniel; Cléry, Antoine; Allain, Frédéric H.‐T.

doi:10.1093/nar/gkab135

Cited by 14 publications

(18 citation statements)

References 77 publications

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“…4A). U1 snRNP was reconstituted using recombinant components (21), and SF3A1-UBL was added before ultraviolet (UV) irradiation to induce protein-RNA cross-linking. The detection of all U1 snRNP proteins and protein-RNA cross-links for U1-70K and SmD2 fit well with the U1 snRNP structure and confirmed the correct reconstitution of the particle (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PTBP1-RRM1 could prevent the contacts of SF3A1 to the UUCG tetraloop and the correct positioning of the UBL core and the carboxyl-terminal tail. Additionally, PTBP1-RRM2, which has also been shown to bind U1-SL4, could increase the affinity of PTBP1 for U1-SL4 and might contribute to the steric hindrance of SF3A1-UBL binding (13,21,27).…”

Section: Discussionmentioning

confidence: 99%

“…GST-SF3A1-UBL for EMSA experiments was prepared as before (16). U1 snRNP in vitro reconstitution was performed as described previously (21).…”

Section: Methodsmentioning

confidence: 99%

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Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly

Vries

Martelly

Campagne

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, the structural basis for the interaction between U1 and U2 small nuclear ribonucleoproteins (snRNPs) during the early steps of splicing is still elusive. The binding of the ubiquitin-like (UBL) domain of SF3A1 to the stem-loop 4 of U1 snRNP (U1-SL4) contributes to this interaction. Here, we determined the 3D structure of the complex between the UBL of SF3A1 and U1-SL4 RNA. Our crystallography, NMR spectroscopy, and cross-linking mass spectrometry data show that SF3A1-UBL recognizes, sequence specifically, the GCG/CGC RNA stem and the apical UUCG tetraloop of U1-SL4. In vitro and in vivo mutational analyses support the observed intermolecular contacts and demonstrate that the carboxyl-terminal arginine-glycine-glycine-arginine (RGGR) motif of SF3A1-UBL binds sequence specifically by inserting into the RNA major groove. Thus, the characterization of the SF3A1-UBL/U1-SL4 complex expands the repertoire of RNA binding domains and reveals the capacity of RGG/RG motifs to bind RNA in a sequence-specific manner.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly

Vries

Martelly

Campagne

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…The results so far have shown that SRSF1 forms a heterodimer with U1 snRNP and is recruited by it to 5′SSs. To test whether the interaction between U1 snRNP and SRSF1 could be direct, we titrated 13 C ILV‐labelled SRSF1∆RS with in vitro reconstituted U1 snRNP using NMR spectroscopy (Fig 5A ), as we did previously for other splicing factors (Campagne et al , 2019 , 2021 ; Jutzi et al , 2020 ). SRSF1∆RS lacks the RS domain (amino acids 198–248) and is more soluble than the full‐length protein.…”

Section: Resultsmentioning

confidence: 99%

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

et al. 2021

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SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5 0 splice site (SS), and both factors affect 5 0 SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5 0 SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.

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“…Additionally, the cat RAPID online database predicted that circIL4R has the potential to bind with several proteins, such as SRSF9 and PTBP1. Previous studies have reported that these two proteins were involved in alternative splicing [ 57 , 58 ]. Whether circIL4R could regulate the alternative splicing via binding with SRSF9 or PTBP1 need further investigation.…”

Section: Discussionmentioning

confidence: 99%

CircIL4R activates the PI3K/AKT signaling pathway via the miR-761/TRIM29/PHLPP1 axis and promotes proliferation and metastasis in colorectal cancer

Jiang

Wang

et al. 2021

Mol Cancer

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Background Accumulating studies have revealed that aberrant expression of circular RNAs (circRNAs) is widely involved in the tumorigenesis and progression of malignant cancers, including colorectal cancer (CRC). Nevertheless, the clinical significance, levels, features, biological function, and molecular mechanisms of novel circRNAs in CRC remain largely unexplored. Methods CRC-related circRNAs were identified through bioinformatics analysis and verified in clinical specimens by qRT–PCR and in situ hybridization (ISH). Then, in vitro and in vivo experiments were performed to determine the clinical significance of, functional roles of, and clinical characteristics associated with circIL4R in CRC specimens and cells. Mechanistically, RNA pull-down, fluorescence in situ hybridization (FISH), luciferase reporter, and ubiquitination assays were performed to confirm the underlying mechanism of circIL4R. Results CircIL4R was upregulated in CRC cell lines and in sera and tissues from CRC patients and was positively correlated with advanced clinicopathological features and poor prognosis. Functional experiments demonstrated that circIL4R promotes CRC cell proliferation, migration, and invasion via the PI3K/AKT signaling pathway. Mechanistically, circIL4R was regulated by TFAP2C and competitively interacted with miR-761 to enhance the expression of TRIM29, thereby targeting PHLPP1 for ubiquitin-mediated degradation to activate the PI3K/AKT signaling pathway and consequently facilitate CRC progression. Conclusions Our findings demonstrate that upregulation of circIL4R plays an oncogenic role in CRC progression and may serve as a promising diagnostic and prognostic biomarker for CRC detection and as a potential therapeutic target for CRC treatment.

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An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands

Cited by 14 publications

References 77 publications

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

CircIL4R activates the PI3K/AKT signaling pathway via the miR-761/TRIM29/PHLPP1 axis and promotes proliferation and metastasis in colorectal cancer

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