An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing

Xu, Deming; Nouraini, Shahrzad; Field, Deborah J.; Tang, Shou‐jiang; Friesen, James D.

doi:10.1038/381709a0

Cited by 77 publications

(85 citation statements)

References 22 publications

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“…One possibility would be that specific RNA substrates exist that remain to be discovered. For example, it has been described that the ATPase activity of protein Slt22p, which has a functional role in pre-mRNA splicing, is stimulated preferentially by annealed U2/U6 small nuclear RNAs (Xu et al, 1996). Among the DEAD-box proteins that have been studied biochemically, only DbpA from E. coli shows a strong RNA substrate specificity (FullerPace et al, 1993).…”

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confidence: 99%

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Zirwes

Eilbracht

Kneissel

et al. 2000

MBoC

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We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ϳ11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ϳ4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit. INTRODUCTIONNucleoli, the most conspicuous intranuclear structures in eukaryotic cells, are known to be the main site of ribosome biosynthesis (Hadjiolov, 1985;Scheer and Weisenberger, 1994;Scheer and Hock, 1999). More recently, however, it has also become clear that the nucleolus has additional functions and may be involved in the transport or assembly of various kinds of ribonucleoprotein particles (reviewed by Pederson, 1998).The production of preribosomal particles is a complex process, involving the transcription of the rRNA genes, the processing of the primary transcripts, and the addition of proteins to the nascent preribosomes as well as the incorporation of the 5S rRNA, which is synthesized outside of the nucleolus. Besides the rRNA gene clusters and flanking sequences, nucleoli contain a large number of proteins and RNA components that are not part of mature cytoplasmic ribosomes but are involved in the transcriptional (Reeder, 1990;Paule, 1993) and post-transcriptional regulation of ribosome synthesis (Olson, 1990). Among these are small nucleolar RNAs (snoRNAs), which play a major role in the endo-and exonucleolytic processing of the large preribosomal precursor as well as in the specific modification of rRNA molecules by remodeling certain uridine residues into pseudouridines and by tagging ribose moieties with methyl groups (To...

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confidence: 99%

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Zirwes

Eilbracht

Kneissel

et al. 2000

MBoC

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“…127 The ATP-dependent loss of U4 and U5 snRNAs was suggested to represent Prp28-mediated displacement of the tri-snRNP. 127 However, the observations that Sad1 knockdown 99 or truncation of the Brr2 NTR 55 lead to non-canonical Brr2-mediated disruption of the tri-snRNP, that NTR truncations lead to increased loss of U5 and U6 snRNAs upon spliceosome activation 44 and that a mutation of a Brr2 residue (E909), which supports the relative domain arrangement in the NC, is associated with the loss of U4 and U5 snRNAs from B complex spliceosomes 45 indicate that the process observed could alternatively represent a Brr2-mediated discard pathway.…”

Section: Brr2 Regulation During Splicingmentioning

confidence: 99%

“…46 Curiously, the E909K exchange in Brr2 blocks splicing in extracts at or before the first catalytic step and leads to the appearance of an off-pathway spliceosomal particle following B complex formation, which lacks U4 and U5 snRNAs. 45 E909 is located in the linker connecting the RecA2 to the WH domains in the NC of Brr2 and apparently stabilizes the relative orientation of the domains similar to the NC-clamp of the NTR (Fig. 2D).…”

Section: Brr2 Regulation During Splicingmentioning

confidence: 99%

“…40,41,52,63,64 Based on the observations that a temperature-sensitive yeast Brr2 variant (E909K), encoded by the slt22-1 allele, is synthetically lethal with mutations in U2 or U6 snRNAs that affect the stability or conformation of U2/U6 helix II, and that the ATPase activity of this variant is no longer stimulated by a U2/ U6 duplex, it was proposed that Brr2 might proofread U2/U6 interactions. 45 Furthermore, different cross-linking profiles of wt Brr2 and a variant exhibiting a residue exchange (G858R) in the NC 5 0 HP/separator loop with U6 snRNA 46 together with the latter variant exhibiting a second step defect and showing synthetic lethality with step 2 mutations in Slu7, Prp18 and Prp16 46,49 suggest that Brr2 participates in a transient opening of the catalytic core between the 2 steps of splicing, which is characterized by the intermittent disruption of U6-5SS and U2-U6 interactions. 125,126 However, whether Brr2 actively disrupts RNA-RNA or RNA-protein interaction in this phase of splicing or whether it simply provides RNA binding sites is presently unclear.…”

Section: Brr2 Regulation During Splicingmentioning

confidence: 99%

“…Brr2, in contrast, stays associated with the spliceosome after recruitment of the tri-snRNP during the remaining phases of splicing. Moreover, in addition to spliceosome activation, Brr2 has been found to be important for the retention of U5 and U6 snRNAs during spliceosome activation 44,45 and to play a role during splicing catalysis 46,47 and spliceosome disassembly. 48 However, not all of these additional functions apparently rely on the enzyme's ATPase and helicase activities.…”

Section: Brr2 Requires Tight Regulationmentioning

confidence: 99%

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Functions and regulation of the Brr2 RNA helicase during splicing

2016

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Pre-mRNA splicing entails the stepwise assembly of an inactive spliceosome, its catalytic activation, splicing catalysis and spliceosome disassembly. Transitions in this reaction cycle are accompanied by compositional and conformational rearrangements of the underlying RNA-protein interaction networks, which are driven and controlled by 8 conserved superfamily 2 RNA helicases. The Ski2-like helicase, Brr2, provides the key remodeling activity during spliceosome activation and is additionally implicated in the catalytic and disassembly phases of splicing, indicating that Brr2 needs to be tightly regulated during splicing. Recent structural and functional analyses have begun to unravel how Brr2 regulation is established via multiple layers of intra-and inter-molecular mechanisms. Brr2 has an unusual structure, including a long N-terminal region and a catalytically inactive C-terminal helicase cassette, which can auto-inhibit and auto-activate the enzyme, respectively. Both elements are essential, also serve as protein-protein interaction devices and the N-terminal region is required for stable Brr2 association with the tri-snRNP, tri-snRNP stability and retention of U5 and U6 snRNAs during spliceosome activation in vivo. Furthermore, a C-terminal region of the Prp8 protein, comprising consecutive RNase H-like and Jab1/MPN-like domains, can both up-and downregulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis-and transregulatory mechanisms. Comparison of isolated Brr2 to electron cryo-microscopic structures of yeast and human U4/U6 U5 tri-snRNPs and spliceosomes indicates how some of the regulatory elements exert their functions during splicing. The various modulatory mechanisms acting on Brr2 might be exploited to enhance splicing fidelity and to regulate alternative splicing. KEYWORDSPre-mRNA splicing; regulation of pre-mRNA splicing; remodeling of RNAprotein complexes; RNA helicase structure, function and regulation; spliceosome catalytic activation

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Small Nuclear Rnps (Snrnps)

Will¹,

Lührmann²

2002

Encyclopedia of Molecular Biology

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An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing

Cited by 77 publications

References 22 publications

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Functions and regulation of the Brr2 RNA helicase during splicing

Small Nuclear Rnps (Snrnps)

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