Biochemical characterization of the ATPase and helicase activity of UAP56, an essential pre-mRNA splicing and mRNA export factor

DEXD/H-box protein UAP56 is an essential pre-mRNA splicing factor required for the first ATP-dependent spliceosome assembly step. UAP56 is also essential for the export of the majority of mRNAs from the nucleus to the cytoplasm. We performed biochemical characterization of UAP56's ATPase and helicase activity, which is important for further understanding the role of these activities in UAP56's function. We showed that UAP56 is an RNA-stimulated ATPase that can only hydrolyze ATP. We demonstrated that UAP56 is an ATP-dependent RNA helicase that can unwind substrates with 5' or 3' overhangs or blunt ends in vitro. We showed that U2AF(65) and Aly, two proteins known to interact with UAP56, do not influence UAP56's ATPase or helicase activity. We also demonstrated that several mutants in the conserved helicase motifs I, II, and III abolish UAP56's ATPase and/or helicase activity, providing tools for future investigation of the role of UAP56's ATPase and helicase activity in spliceosome assembly and mRNA export.     

The nuclear export signal of splicing factor Uap56p interacts with nuclear pore-associated protein Rae1p for mRNA export in Schizosaccharomyces pombe

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.     

mRNA export: travelling with DEAD box proteins.

Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene.     

The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila.

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.     

Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56

URH49 is a mammalian protein that is 90% identical to the DExH/D box protein UAP56, an RNA helicase that is important for splicing and nuclear export of mRNA. Although Saccharomyces cerevisiae and Drosophila express only a single protein corresponding to UAP56, mRNAs encoding URH49 and UAP56 are both expressed in human and mouse cells. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), indicating that both proteins have similar functions. UAP56 mRNA is more abundant than URH49 mRNA in many tissues, although in testes URH49 mRNA is much more abundant. UAP56 and URH49 mRNAs are present at similar levels in proliferating cultured cells. However, when the cells enter quiescence, the URH49 mRNA level decreases 3–6-fold while the UAP56 mRNA level remains relatively constant. The amount of URH49 mRNA increases to the level found in proliferating cells within 5 h when quiescent cells are growth-stimulated or when protein synthesis is inhibited. URH49 mRNA is relatively unstable (T_½ = 4 h) in quiescent cells, but is stabilized immediately following growth stimulation or inhibition of protein synthesis. In contrast, there is much less change in the content or stability of UAP56 mRNA following growth stimulation. Our observations suggest that in mammalian cells, two UAP56-like RNA helicases are involved in splicing and nuclear export of mRNA. Differential expression of these helicases may lead to quantitative or qualitative changes in mRNA expression.     

Interferon-induced antiviral protein MxA interacts with the cellular RNA helicases UAP56 and URH49

Mx proteins are a family of large GTPases that are induced exclusively by interferon-α/β and have a broad antiviral activity against several viruses, including influenza A virus (IAV). Although the antiviral activities of mouse Mx1 and human MxA have been studied extensively, the molecular mechanism of action remains largely unsolved. Because no direct interaction between Mx proteins and IAV proteins or RNA had been demonstrated so far, we addressed the question of whether Mx protein would interact with cellular proteins required for efficient replication of IAV. Immunoprecipitation of MxA revealed its association with two closely related RNA helicases, UAP56 and URH49. UAP56 and its paralog URH49 play an important role in IAV replication and are involved in nuclear export of IAV mRNAs and prevention of dsRNA accumulation in infected cells. In vitro binding assays with purified recombinant proteins revealed that MxA formed a direct complex with the RNA helicases. In addition, recombinant mouse Mx1 was also able to bind to UAP56 or URH49. Furthermore, the complex formation between cytoplasmic MxA and UAP56 or URH49 occurred in the perinuclear region, whereas nuclear Mx1 interacted with UAP56 or URH49 in distinct dots in the nucleus. Taken together, our data reveal that Mx proteins exerting antiviral activity can directly bind to the two cellular DExD/H box RNA helicases UAP56 and URH49. Moreover, the observed subcellular localization of the Mx-RNA helicase complexes coincides with the subcellular localization, where human MxA and mouse Mx1 proteins act antivirally. On the basis of these data, we propose that Mx proteins exert their antiviral activity against IAV by interfering with the function of the RNA helicases UAP56 and URH49.     

UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.     

Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module

MLN51 is a nucleocytoplasmic shuttling protein that is overexpressed in breast cancer. The function of MLN51 in mammals remains elusive. Its fly homolog, named barentsz, as well as the proteins mago nashi and tsunagi have been shown to be required for proper oskar mRNA localization to the posterior pole of the oocyte. Magoh and Y14, the human homologs of mago nashi and tsunagi, are core components of the exon junction complex (EJC). The EJC is assembled on spliced mRNAs and plays important roles in post-splicing events including mRNA export, nonsense-mediated mRNA decay, and translation. In the present study, we show that human MLN51 is an RNA-binding protein present in ribonucleo-protein complexes. By co-immunoprecipitation assays, endogenous MLN51 protein is found to be associated with EJC components, including Magoh, Y14, and NFX1/TAP, and subcellular localization studies indicate that MLN51 transiently co-localizes with Magoh in nuclear speckles. Moreover, we demonstrate that MLN51 specifically associates with spliced mRNAs in co-precipitation experiments, both in the nucleus and in the cytoplasm, at the position where the EJC is deposited. Most interesting, we have identified a region within MLN51 sufficient to bind RNA, to interact with Magoh and spliced mRNA, and to address the protein to nuclear speckles. This conserved region of MLN51 was therefore named SELOR for speckle localizer and RNA binding module. Altogether our data demonstrate that MLN51 associates with EJC in the nucleus and remains stably associated with mRNA in the cytoplasm, suggesting that its overexpression might alter mRNA metabolism in cancer.     

PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.     

Direct observation of regulated ribonucleoprotein transport across the nurse cell/oocyte boundary

In Drosophila, the asymmetric localization of specific mRNAs to discrete regions within the developing oocyte determines the embryonic axes. The microtubule motors dynein and kinesin are required for the proper localization of the determinant ribonucleoprotein (RNP) complexes, but the mechanisms that account for RNP transport to and within the oocyte are not well understood. In this work, we focus on the transport of RNA complexes containing bicoid (bcd), an anterior determinant. We show in live egg chambers that, within the nurse cell compartment, dynein actively transports green fluorescent protein-tagged Exuperantia, a cofactor required for bcd RNP localization. Surprisingly, the loss of kinesin I activity elevates RNP motility in nurse cells, whereas disruption of dynein activity inhibits RNP transport. Once RNPs are transferred through the ring canal to the oocyte, they no longer display rapid, linear movements, but they are distributed by cytoplasmic streaming and gradually disassemble. By contrast, bcd mRNA injected into oocytes assembles de novo into RNP particles that exhibit rapid, dynein-dependent transport. We speculate that after delivery to the oocyte, RNP complexes may disassemble and be remodeled with appropriate accessory factors to ensure proper localization.     

RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA

The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.     

Molecular Modeling of the Plasmodium falciparum Pre-mRNA Splicing and Nuclear Export Factor PfU52

UAP56/SUB2 is a DExD/H-box RNA helicase that is critically involved in pre-mRNA splicing and mRNA nuclear export. This helicase is broadly conserved and essential in many eukaryotic lineages, including protozoan and metazoan parasites. Previous research suggests that helicases from parasites could be promising drug targets for treating parasitic diseases. Accordingly, characterizing the structure and function of these proteins is of interest for structure-based, de novo design of new lead compounds. Here, we used homology modeling to construct a three-dimensional structure of PfU52 (PMDB ID: PM0079288), the Plasmodium falciparum ortholog of UAP56/SUB2, and explored the detailed architecture of its functional sites. Comparative in silico analysis revealed that although PfU52 shared many physicochemical, structural and dynamic similarities with its human homolog, it also displayed some unique features that could be exploited for drug design.