Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome

Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Genes & Development (Impact Factor: 10.8). 08/2008; 22(13):1796-803. DOI: 10.1101/gad.1657308
Source: PubMed


The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56's ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF(65) and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56's dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF(65). Our results indicate that hUAP56 first interacts with U2AF(65) in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.

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Available from: Lingdi Zhang, Oct 13, 2014
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    • "In HeLa cell extracts, the SR-related proteins Srm160 and Srm300 have also been shown to bridge the U1 and U2 snRNPs (Eldridge et al. 1999; Blencowe et al. 2000). Finally, in yeast and humans, the UAP56 protein was shown to facilitate the stable ATP-dependent contact of the U2 snRNP with the branch point sequence, but its role in cross-intron bridging is not known (Fleckner et al. 1997; Kistler and Guthrie 2001; Libri et al. 2001; Shen et al. 2008). A recent yeast study reported genetic interactions between the U1 snRNA and the branch point-binding complex of Msl5 (branch point-binding protein or SF1 in humans) and Mud2 (U2AF65 in humans) (Schwer et al. 2013). "
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    ABSTRACT: The pairing of 5' and 3' splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 5' splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3' splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5' and 3' splice site complexes within the assembling spliceosome. © 2014 Sharma et al.; Published by Cold Spring Harbor Laboratory Press.
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    • "U2AF65 recruits UAP56 to the mRNA, where it is required for stable interaction between U2 snRNP and the pre-mRNA branch point [4]. Subsequently, it was found that UAP56 and its yeast counterpart termed SUB2 are involved in several steps of spliceosome assembly and their ATPase and helicase activities are required for that [5], [6]. Biochemical characterisation of human UAP56 revealed that it has RNA-stimulated ATPase activity and ATP-dependent RNA helicase activity [7]. "
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    ABSTRACT: The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.
    Full-text · Article · Mar 2013 · PLoS ONE
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    • "UAP56 is an ATP dependent RNA helicase with ATPase activity that is a member of the DExD/H box family of RNA helicases [7]. Like other DExD/H box proteins, UAP56 plays an important role in several steps of RNA synthesis and function including RNA splicing and mRNA transport from the nucleus to the cytoplasm [8] [9] [10]. Yamazaki et al. have shown that UAP56 forms an mRNA export machinery that regulates mitotic progression [11]. "
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    ABSTRACT: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.
    Full-text · Article · Dec 2012 · Biochemical and Biophysical Research Communications
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