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

Abstract

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. Pre-mRNA splicing occurs in a ribonucleoprotein (RNP) complex called the spliceosome, which comprises a large number of proteins and several U small nuclear RNP particles (snRNPs) termed U1, U2, U4, U5, and U6. Assembly of the spliceosome on the pre-mRNA substrate proceeds in a stepwise manner and involves the recognition of intron-defining splice signals (for review, see Hertel and Graveley 2005). Spliceosome assembly is initiated by the formation of complex E, in which U1 snRNP is stably associated with the 5Ј splice site, SF1 is associated with the branchpoint, and U2 snRNP auxiliary factor (U2AF) subunits U2AF 65 and U2AF 35 are associated with the polypyrimidine (Py)-tract and 3Ј splice site, respectively. Subsequently, SF1 is replaced by U2 snRNP at the branchpoint, leading to the formation of complex A (the prespliceosome). Mature spliceosome assembly occurs upon entry of the U4/U6 и U5 tri-snRNP to form complex B, followed by structural rearrangements to form the catalytically active complex C, in which U2 and U6 snRNAs interact, and U6 replaces U1 at the 5Ј splice site.Many steps in spliceosome assembly require ATP hydrolysis and are mediated by a series of splicing factors that are members of the DExD/H-box protein family, the founding member of which is eIF-4A, a known RNA helicase (for review, see Staley and Guthrie 1998;Silverman et al. 2003;Rocak and Linder 2004). Several of these DExD/H-box splicing factors have been shown to possess an ATP-dependent RNA unwinding/helicase activity (Laggerbauer et al. 1998;Raghunathan and Guthrie 1998;Wagner et al. 1998;Wang et al. 1998) and are thought to use ATP hydrolysis as a driving force to modulate specific RNA structural rearrangements during spliceosome assembly. DExD/H-box proteins typically have a series of conserved sequence motifs; structural, mutational, and biochemical analyses have suggested roles for these motifs in ATP-binding, ATP hydrolysis (ATPase), RNA-binding, and dsRNA unwinding/hel...

From unwinding to clamping — the DEAD box RNA helicase family

“…Other emerging themes are exemplified by the proposed functions of the DEAD box helicase Sub2 (UAP56 in vertebrates) in promoting protein-protein interactions in an ATP-dependent manner, a function that is important for the assembly of spliceosomes on mRNAs 131 . The involvement of DEAD box proteins, such as Dbp4, in the release of snoRNAs from the preribosome is another example of an important way in which RNPs can be modulated 132 .…”

Proteomics of tissue factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control

“…UAP56 is an important factor for spliceosome assembly, mRNA export and translation, and 390 it plays an important role in cell growth and proliferation [36][37][38]. DDX39, a paralogue of 391 UAP56 that is expressed in a growth-regulated manner [39,40], was also down-regulated after 392 TF silencing.…”

Influenza A Virus Multiplication and the Cellular SUMOylation System

“…Moreover, in vitro analysis of viral transcription revealed about a ~5-fold reduction in the fraction of viral polyadenylated (positive sense) transcripts, suggesting that SFPQ/PSF seems to facilitate the polyadenylation of viral transcripts [113]. UAP56, (Bat1/Raf-2p48), a known viral-interactor protein that was also identified in a vRdRp interaction screening by Mayer et al [89], is a fairly well established RNA helicase involved in spliceosome assembly [114], facilitating the nuclear export of cellular mRNA [115], and is a constituent of the transcription export complex which delivers pre-mRNAs bound to the exon-junction complex to nuclear export factor 1 (NXF1) [116]. Although UAP56 is clearly a factor in cellular RNA splicing, in vitro studies indicate that UAP56 forms heterodimers with NP in the absence of vRNA.…”