The nuclear basket mediates perinuclear mRNA scanning in budding yeast

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.     

Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export

The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome‐mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap‐binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.     

Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment

The deposition of proteins onto newly spliced mRNAs has far reaching consequences for their subsequent metabolism. We affinity-purified spliced human mRNPs under physiological conditions from HeLa nuclear extract and present the first comprehensive inventory of their protein composition as determined by mass spectrometry. Several proteins previously not known to be mRNP-associated were detected, including the DEAD-box helicases DDX3, DDX5, and DDX9, and the ELG, hNHN1, BCLAF1, and TRAP150 proteins. The association of some of the newly identified mRNP proteins was shown to be splicing-dependent, but not to require EJC formation. Initial recruitment of EJC proteins to the spliceosome did not require an EJC binding platform at the -20/24 region of the 5' exon. Finally, while recruitment of EJC proteins and stable EJC formation were not dependent on the cap binding complex, several of the newly identified mRNP proteins required the latter for their association with mRNPs. These results provide novel insights into the composition of spliced mRNPs and the requirements for the association of mRNP proteins with the newly spliced mRNA.     

URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway

The TREX complex integrates information from nuclear mRNA processing events to ensure the timely export of mRNA to the cytoplasm. In humans, UAP56 and its paralog URH49 form distinct complexes, the TREX complex and the AREX complex, respectively, which cooperatively regulate the expression of a specific set of mRNA species on a genome wide scale. The difference in the complex formation between UAP56 and URH49 are thought to play a critical role in the regulation of target mRNAs. To date, the underlying mechanism remains poorly understood. Here we characterize the formation of the TREX complex and the AREX complex. In the ATP depleted condition, UAP56 formed an Apo-TREX complex containing the THO subcomplex but not ALYREF and CIP29. URH49 formed an Apo-AREX complex containing CIP29 but not ALYREF and the THO subcomplex. However, with the addition of ATP, both the Apo-TREX complex and the Apo-AREX complex were remodeled to highly similar ATP-TREX complex containing the THO subcomplex, ALYREF and CIP29. The knockdown of URH49 caused a reduction in its target mRNAs and a cytokinesis failure. Similarly, cytokinesis abnormality was observed in CIP29 knockdown cells, suggesting that CIP29 belongs to the URH49 regulated mRNA export pathway. Lastly, we confirmed that the export of mRNA in URH49-dependent pathway is achieved by NXF1, which is also observed in UAP56-dependent pathway. Our studies propose an mRNA export model that the mRNA selectivity depends on the Apo-form TREX/AREX complex, which is remodeled to the highly similar ATP-form complex upon ATP loading, and integrated to NXF1.     

Human mRNA export machinery recruited to the 5' end of mRNA

Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.     

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.     

The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions

Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half-life. There is accumulating evidence that pre-mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of approximately 335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20-24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed.     

An RNA‐binding atypical tropomyosin recruits kinesin‐1 dynamically to oskar mRNPs

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.     

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.     

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.     

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.     

REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon-exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, we show that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, we conclude that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. Our results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1.     

U2AF participates in the binding of TAP (NXF1) to mRNA.

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates.