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ICLIP and NGS to map protein-RNA interactions for U1-snRNP proteins in Trypanosoma brucei

ABSTRACT: Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei.

ORGANISM(S): Trypanosoma brucei

PROVIDER: GSE43848 | GEO | 2014/05/16

SECONDARY ACCESSION(S): PRJNA187579

REPOSITORIES: GEO

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iCLIP and NGS to map protein-RNA interactions for U1-snRNP proteins in Trypanosoma brucei

Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei. 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking

2014-05-16 | E-GEOD-43848 | biostudies-arrayexpress

The U1 snRNP protein U1C and Helix H of U1 snRNA are critical for small molecule splicing modulator function

Project description:Risdiplam and branaplam are two small molecule splicing modulators that enhance the ability of U1 snRNP to recognize non-canonical GA/GU-containing 5’ splice sites (ss). We demonstrated that branaplam improves the recognition of these 5’ ss by reconstituted U1 snRNP in vitro and that this effect is dependent on the ZnF domain of U1C and Helix H of U1 snRNA but not U1A or U1-70K. We then showed in vivo depletion of U1C decreases the potency of most compound-induced cassette exons. However, many cassette exons only become responsive to compound when U1C is depleted, leading to a model where U1C stabilizes different context-dependent structural conformations of U1 snRNP which either promotes or hinders compound binding and its effect on splicing. Surprisingly, risdiplam has no effect on the recognition of these 5’ ss in vitro, suggesting additional cellular factors are required.

2026-07-24 | GSE304951 | GEO

U1 snRNP and RNA polymerase II interaction is predominantly mediated by Prp40 rather than U1-70K in yeast

Project description:Transcription and splicing are coupled both temporally and physically. A previous cryo-EM structure of the human U1 snRNP and RNA polymerase pol II complex has shown that U1 snRNP uses predominantly the RRM domain of U1-70K to directly interact with the RPB2 subunit of pol II. However, residues on U1-70K involved in the interaction with pol II are not conserved in yeast U1-70K, raising the question whether yeast U1 snRNP interacts with pol II in a similar manner. We found that yeast pol II associates with both U1 and U2 snRNPs, but U1-70K makes a minimal contribution to U1 snRNP’s interaction with pol II. On the other hand, multiple domains of yeast Prp40 interact with pol II and the removal of the C-terminal domain (CTD) of pol II does not affect this interaction. Although yeast Prp40 is stably associated with U1 snRNP, its human homologs, PRPF40a and PRPF40b, are alternative splicing factors that are not integral components of U1 snRNP. This shift of function of Prp40 homologs may have led to the evolution of U1-70K to be the main interactor with pol II in the human system.

2026-05-20 | PXD078573 | Pride

poly(A+) RNAs 3'-seq analysis in control and U1-specific proteins KD (knock-down) samples

Project description:To understand the effect of U1 snRNP-specific proteins on poly(A+) RNAs profiles, we carried out control and U1A, U1C, U1-70k depletion (using synthetic siRNAs) experiments followed by poly(A+) RNA 3'-seq anlyais using lexogen kit (016.024)in HeLa cells

2023-02-27 | GSE192820 | GEO

Alzheimer’s disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment [N40K neuron RNA-seq]

Project description:Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, neuronal degeneration, cognitive impairment, and erroneous splicing events in synaptic pathways.

2022-08-11 | GSE196872 | GEO

Alzheimer’s disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment [N40K mice RNA-seq]

2022-08-11 | GSE196871 | GEO

Mus musculus

Project description:tRIP for U1A/U1-70K/U1C, U1C-FUS and RNAP II-U1C

| PRJDB8097 | ENA

A U1-70K Protein Interaction Network Reveals a Functional Class of RNA Binding Proteins with Mixed Charge Domains

Project description:U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other proteins of the spliceosome complex are mislocalized to cytoplasmic aggregates in Alzheimer’s disease (AD) brain, yet understanding of the specific mechanisms that cause their aggregation is limited. Many of the RNA binding proteins (RBPs) that aggregate in neurodegenerative diseases, including TDP-43 and FUS, self-assemble into RNA granules through disordered low complexity (LC) domains. We report here that a LC domain within U1-70K, that is comprised of tandem arrays of basic (R/K) and acidic (D/E) residues, shares many of the same properties of the Q/N-rich prion-like LC domains found in TDP-43 and FUS. These properties include the ability to self-assemble into oligomers, and to form nuclear granules in cells. To analyze the functional roles of the U1-70K LC domains, we performed co-immunoprecipitation of recombinant U1-70K and deletions lacking the C-terminal LC domain(s) followed by quantitative proteomics. Using a network-driven approach, functional classes of U1-70K interacting proteins were identified that showed a varying dependency on the U1-70K LC domain(s) for their interaction. We characterize a family of structurally homologous RBPs containing U1-70K LC1-like domains including LUC7L3 and RBM25, which require their respective LC1-like domains for reciprocal interactions with U1-70K and for participation in nuclear RNA granules. Finally, we also show that the LC1 domain of U1-70K can interact with Tau from AD brain supporting a hypothesis that mixed charge structural motifs on U1-70K and other RBPs could mediate cooperative interactions with pathological tau isoforms

2018-06-21 | PXD008260 | Pride

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

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.

2013-06-28 | E-GEOD-46433 | biostudies-arrayexpress

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