Homodimerization of the human U1 snRNP-specific protein C

Gunnewiek, Jacqueline M. T. Klein; Aarssen, Yvonne van; Wassenaar, R.; Legrain, Pierre; Venrooij, W.J. van; Nelissen, R.L.H.

doi:10.1093/nar/23.23.4864

Cited by 23 publications

(15 citation statements)

References 32 publications

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“…KREPB2 was identified as an editosome component by mass spectrometry, and found to contain an RNase III motif harboring amino acids that are critical for catalysis, a U1 Zn 2ϩ finger, and double-stranded RNA binding. It has sequence similarity to KREN1 and KREN2, which also contain these three motifs (23,41,64). These data strongly suggest an RNA editing endonuclease role for KREPB2.…”

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confidence: 69%

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Carnes

Trotter

Peltan

et al. 2008

Molecular and Cellular Biology

106

150

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Trypanosoma brucei has three distinct ϳ20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ϳ20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.RNA editing recodes most of the mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridine nucleotides (U's) using information provided by guide RNAs (gRNAs) (58). RNA editing is developmentally regulated and is required for parasites that cycle between insect vector and mammalian host (54). Proteins that perform catalytic steps of RNA editing have been identified: endonucleases KREN1 or KREN2 cleave at deletion or insertion sites, respectively; terminal uridylyl transferase (TUTase) KRET2 adds U's at insertion sites; U specific exoribonuclease (exoUase) KREX1 removes U's at deletion sites; and ligases KREL1 or KREL2 rejoin mRNA fragments after U addition or removal (5,13,25,30,34,55,61). These catalytic steps are coordinated by the multiprotein editosomes that sediment at ϳ20S on glycerol gradients (9, 43). KREPB2 was identified as an editosome component by mass spectrometry, and found to contain an RNase III motif harboring amino acids that are critical for catalysis, a U1 Zn 2ϩ finger, and double-stranded RNA binding. It has sequence similarity to KREN1 and KREN2, which also contain these three motifs (23,41,64). These data strongly suggest an RNA editing endonuclease role for KREPB2.KREN1 and KREN2 were shown to be RNA editing endonucleases using both RNA interference (RNAi) and conditional knockout cell lines (5, 61). Both KREN1 and KREN2 are essential for the normal growth of PF and BF cells, and repression of their expression by either method led to dramatic growth defects or death, respectively. Such repression of KREN1 eliminated in vitro cleavage by ϳ20S editosomes of a deletion site in a synthetic substrate modeled on ATPase subunit 6 (A6) pre-mRNA but did not alter cleavage of an insertion site in a substrate derived from the same mRNA. These studies and other data have shown that KREN1 is an editing endonuclease with a preference for sites from which U's are deleted (29). Similarly, repression of KREN2 eliminated in vitro cleavage at insertion ed...

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mentioning

confidence: 69%

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Carnes

Trotter

Peltan

et al. 2008

Molecular and Cellular Biology

106

150

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“…The U1-C protein could not be recognized as an individual domain. Although these results suggested the presence of one copy of U1-C, other biochemical experiments suggested that U1-C in the U1 snRNP could be present as a dimer (17).…”

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confidence: 76%

Protein Stoichiometry of a Multiprotein Complex, the Human Spliceosomal U1 Small Nuclear Ribonucleoprotein

Hochleitner

Kastner

Fröhlich

et al. 2005

Journal of Biological Chemistry

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The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

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“…There is evidence that U1C is required for stable interaction between U1 snRNP and the pre-mRNA 5= ss (17). U1C carries a cystidine/histidine zinc finger-like motif at its N terminus, which is necessary and sufficient for its binding to the U1 snRNP (32) as well as for homodimerization (15). The zinc finger-like motif containing region is required for U1C interaction with RBFOX2.…”

Section: Discussionmentioning

confidence: 99%

RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

Huang

Park

et al. 2012

Molecular and Cellular Biology

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The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5= splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5= splice site, but not the engineered strong 5= splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5= splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5= splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5= splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.A lternative splicing is a eukaryotic regulatory mechanism that allows for the generation of numerous protein isoforms with often diverse biological functions from a single gene (4,26,41). It begins with the spliceosome, which is assembled stepwise by the addition of discrete small nuclear ribonucleoprotein particles (snRNPs) and numerous accessory non-snRNP splicing factors (23, 33). The excision of introns followed by the joining of exons depends on the recognition and usage of 5= and 3= splice sites (5= ss and 3= ss, respectively) by the splicing machinery (19, 34). The initial splicing step is comprised of 5= ss recognition by U1 snRNP and binding of U2 auxiliary factor (U2AF) to the 3= ss. These factors and additional proteins form the E or commitment complex, which bridges the intron and brings the splice sites close together. U2AF then recruits U2 snRNP to form the A complex. Subsequent binding of the U4-U6-U5 tri-snRNP and many other factors result in a fully assembled spliceosome that supports a series of rearrangements via RNA-RNA and RNA-protein interactions and activates the catalytic steps of cleavage, exon joining, and intron release (4, 26).The splice site signals that define the 5= ss and 3= ss of an alternatively spliced exon are often weak. How and when they are used is believed to be modulated by a complex interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (4, 26). These form the basis for alternative splicing. Target prediction for specific splicing factors is difficult, largely due to the small size and degeneracy of splicing factorbinding motifs. An exception to this degeneracy is the hexanucleotide UGCAUG, which has been shown to be an important element for the splicing of several exons (3,5,14,16,20,24,...

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Homodimerization of the human U1 snRNP-specific protein C

Cited by 23 publications

References 32 publications

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Protein Stoichiometry of a Multiprotein Complex, the Human Spliceosomal U1 Small Nuclear Ribonucleoprotein

RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

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