The RNA helicase DDX39 contributes to the nuclear export of spliceosomal U snRNA by loading of PHAX onto RNA.

RNA helicases are involved in RNA metabolism in an ATP-dependent manner. Although many RNA helicases unwind the RNA structure and/or remove proteins from the RNA, some can load their interacting proteins onto RNAs. Here, we developed an in vitro strategy to identify the ATP-dependent factors involved in spliceosomal uridine-rich small nuclear RNA (U snRNA) export. We identified the RNA helicase UAP56/DDX39B, a component of the mRNA export complex named the transcription-export (TREX) complex, and its closely related RNA helicase URH49/DDX39A as the factors that stimulated RNA binding of PHAX, an adapter protein for U snRNA export. ALYREF, another TREX component, acted as a bridge between PHAX and UAP56/DDX39B. We also showed that UAP56/DDX39B and ALYREF participate in U snRNA export through a mechanism distinct from that of mRNA export. This study describes a novel aspect of the TREX components for U snRNP biogenesis and highlights the loading activity of RNA helicases.

The hnRNP C tetramer binds to CBC on mRNA and impedes PHAX recruitment for the classification of RNA polymerase II transcripts.

In eukaryotic cells, various classes of RNAs are exported to the cytoplasm by class-specific factors. Accumulating evidence has shown that export factors affect the fate of RNA, demonstrating the importance of proper RNA classification upon export. We previously reported that RNA polymerase II transcripts were classified after synthesis depending on their length, and identified heterogeneous nuclear ribonucleoprotein (hnRNP) C as the key classification factor. HnRNP C inhibits the recruitment of PHAX, an adapter protein for spliceosomal U snRNA export, to long transcripts, navigating these RNAs to the mRNA export pathway. However, the mechanisms by which hnRNP C inhibits PHAX recruitment to mRNA remain unknown. We showed that the cap-binding complex, a bridging factor between m7G-capped RNA and PHAX, directly interacted with hnRNP C on mRNA. Additionally, we revealed that the tetramer-forming activity of hnRNP C and its strong RNA-binding activity were crucial for the inhibition of PHAX binding to longer RNAs. These results suggest that mRNA is wrapped around the hnRNP C tetramer without a gap from the cap, thereby impeding the recruitment of PHAX. The results obtained on the mode of length-specific RNA classification by the hnRNP C tetramer will provide mechanistic insights into hnRNP C-mediated RNA biogenesis.

Structural insight into Marburg virus nucleoprotein-RNA complex formation.

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex constitutes the core structure for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for helical assembly and subsequent viral RNA synthesis. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

ISG20 and nuclear exosome promote destabilization of nascent transcripts for spliceosomal U snRNAs and U1 variants.

Primary RNA transcripts are processed in a plethora of ways to become mature functional forms. In one example, human spliceosomal U snRNAs are matured at their 3'-end by an exonuclease termed TOE1. This process is important because mutations in TOE1 gene can cause a human genetic disease, pontocerebellar hypoplasia (PCH). Nevertheless, TOE1 may not be the only maturation exonuclease for U snRNAs in the cell. Here, we biochemically identify two exonucleolytic factors, Interferon-stimulated gene 20-kDa protein (ISG20) and the nuclear exosome as such candidates, using a newly developed in vitro system that recapitulates 3'-end maturation of U1 snRNA. However, extensive 3'-end sequencing of endogenous U1 snRNA of the knockdown (KD) cells revealed that these factors are not the maturation factors per se. Instead, the nascent transcripts of the spliceosomal U snRNAs as well as of unstable U1 variants were found to increase in quantity upon KD of the factors. These results indicated that ISG20 and the nuclear exosome promote the degradation of nascent spliceosomal U snRNAs and U1 variants, and therefore implied their role in the quality control of newly synthesized U snRNAs.

The RNA transport factor PHAX is required for proper histone H2AX expression and DNA damage response.

PHAX (phosphorylated adaptor for RNA export) promotes nuclear export of short transcripts of RNA polymerase II such as spliceosomal U snRNA precursors, as well as intranuclear transport of small nucleolar RNAs (snoRNAs). However, it remains unknown whether PHAX has other critical functions. Here we show that PHAX is required for efficient DNA damage response (DDR) via regulation of phosphorylated histone variant H2AX (γH2AX), a key factor for DDR. Knockdown of PHAX led to a significant reduction of H2AX mRNA levels, through inhibition of both transcription of the H2AX gene and nuclear export of H2AX mRNA, one of the shortest mRNAs in the cell. As a result, PHAX-knockdown cells become more sensitive to DNA damage due to a shortage of γH2AX. These results reveal a novel function of PHAX, which secures efficient DDR and hence genome stability.

ARS2 Regulates Nuclear Paraspeckle Formation through 3'-End Processing and Stability of NEAT1 Long Noncoding RNA.

Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long noncoding RNA that functions as an essential framework of subnuclear paraspeckle bodies. Of the two isoforms (NEAT1_1 and NEAT1_2) produced by alternative 3'-end RNA processing, the longer isoform, NEAT1_2, plays a crucial role in paraspeckle formation. Here, we demonstrate that the 3'-end processing and stability of NEAT1 RNAs are regulated by arsenic resistance protein 2 (ARS2), a factor interacting with the cap-binding complex (CBC) that binds to the m7G cap structure of RNA polymerase II transcripts. The knockdown of ARS2 inhibited the association between NEAT1 and mammalian cleavage factor I (CFIm), which produces the shorter isoform, NEAT1_1. Furthermore, the knockdown of ARS2 led to the preferential stabilization of NEAT1_2. As a result, NEAT1_2 RNA levels were markedly elevated in ARS2 knockdown cells, leading to an increase in the number of paraspeckles. These results reveal a suppressive role for ARS2 in NEAT1_2 expression and the subsequent formation of paraspeckles.

Exportin-5 mediates nuclear export of SRP RNA in vertebrates.

The signal recognition particle is a ribonucleoprotein complex that is essential for the translocation of nascent proteins into the endoplasmic reticulum. It has been shown that the RNA component (SRP RNA) is exported from the nucleus by CRM1 in the budding yeast. However, how SRP RNA is exported in higher species has been elusive. Here, we show that SRP RNA does not use the CRM1 pathway in Xenopus oocytes. Instead, SRP RNA uses the same export pathway as pre-miRNA and tRNA as showed by cross-competition experiments. Consistently, the recombinant Exportin-5 protein specifically stimulated export of SRP RNA as well as of pre-miRNA and tRNA, whereas an antibody raised against Exportin-5 specifically inhibited export of the same RNA species. Moreover, biotinylated SRP RNA can pull down Exportin-5 but not CRM1 from HeLa cell nuclear extracts in a RanGTP-dependent manner. These results, taken together, strongly suggest that the principal export receptor for SRP RNA in vertebrates is Exportin-5 unlike in the budding yeast.

Analysis of RNA transport in Xenopus oocytes and mammalian cells.

In eukaryotes, many RNA species are transcribed, processed in the nucleus, and exported to the cytoplasm, where they are destined to function or to be further matured. Some RNAs are even reimported to the nucleus. In addition, many RNAs are localized at specific nuclear bodies before their export and/or after their nuclear reimport. To understand how RNAs are transported, Xenopus oocytes are extremely useful cells, thanks to their large size. RNA transport can be easily examined by microinjecting radioactively or fluorescently labeled RNAs into Xenopus oocytes. Mammalian cultured cells are sometimes useful by virtue of RNA-FISH technique. Here, we describe methods to analyze RNA localization and export using these cells.

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment.

Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

Mechanistic insights into human pre-mRNA splicing of human ultra-short introns: potential unusual mechanism identifies G-rich introns.

It is unknown how very short introns (<65 nt; termed 'ultra-short' introns) could be spliced in a massive spliceosome (>2.7 MDa) without steric hindrance. By screening an annotated human transcriptome database (H-InvDB), we identified three model ultra-short introns: the 56-nt intron in the HNRNPH1 (hnRNP H1) gene, the 49-nt intron in the NDOR1 (NADPH dependent diflavin oxidoreductase 1) gene, and the 43-nt intron in the ESRP2 (epithelial splicing regulatory protein 2) gene. We verified that these endogenous ultra-short introns are spliced, and also recapitulated this in cultured cells transfected with the corresponding mini-genes. The splicing of these ultra-short introns was repressed by a splicing inhibitor, spliceostatin A, suggesting that SF3b (a U2 snRNP component) is involved in their splicing processes. The 56-nt intron containing a pyrimidine-rich tract was spliced out in a lariat form, and this splicing was inhibited by the disruption of U1, U2, or U4 snRNA. In contrast, the 49- and 43-nt introns were purine-rich overall without any pyrimidine-rich tract, and these lariat RNAs were not detectable. Remarkably, shared G-rich intronic sequences in the 49- and 43-nt introns were required for their splicing, suggesting that these ultra-short introns may recruit a novel auxiliary splicing mechanism linked to G-rich intronic splicing enhancers.

hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts for export.

Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.

Multiple factors in the early splicing complex are involved in the nuclear retention of pre-mRNAs in mammalian cells.

Intron-containing pre-mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre-mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre-mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre-mRNAs in mammalian cells. By focusing on factors that bind to the 3'-splice site region, we found that the 65-kD subunit of U2AF (U2AF(65) ) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD-box RNA helicase involved in both RNA splicing and export, cooperates with U2AF(65) in exerting nuclear retention activity. Our findings provide new information regarding the pre-mRNA nuclear retention factors in mammalian cells.

U1-independent pre-mRNA splicing contributes to the regulation of alternative splicing.

U1 snRNP plays a crucial role in the 5' splice site recognition during splicing. Here we report the first example of naturally occurring U1-independent U2-type splicing in humans. The U1 components were not included in the pre-spliceosomal E complex formed on the human F1gamma (hF1gamma) intron 9 in vitro. Moreover, hF1gamma intron 9 was efficiently spliced even in U1-disrupted Xenopus oocytes as well as in U1-inactivated HeLa nuclear extracts. Finally, hF1gamma exon 9 skipping induced by an alternative splicing regulator Fox-1 was impaired when intron 9 was changed to the U1-dependent one. Our results suggest that U1-independent splicing contributes to the regulation of alternative splicing of a class of pre-mRNAs.

ATP-dependent recruitment of export factor Aly/REF onto intronless mRNAs by RNA helicase UAP56.

Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

Role of purine-rich exonic splicing enhancers in nuclear retention of pre-mRNAs.

Intron-containing pre-mRNAs are normally retained in the nucleus until they are spliced to produce mature mRNAs that are exported to the cytoplasm. Although the detailed mechanism is not well understood, the formation of splicing-related complexes on pre-mRNAs is thought to be responsible for the nuclear retention. Therefore, pre-mRNAs containing suboptimal splice sites should tend to leak out to the cytoplasm. Such pre-mRNAs often contain purine-rich exonic splicing enhancers (ESEs) that stimulate splicing of the adjacent intron. Here, we show that ESEs per se possess an activity to retain RNAs in the nucleus through a saturable nuclear retention factor. Cross-competition experiments revealed that intron-containing pre-mRNAs (without ESEs) used the same saturable nuclear retention factor as ESEs. Interestingly, although intronless mRNAs containing ESEs were also poorly exported, spliced mRNAs produced from ESE-containing pre-mRNAs were efficiently exported to the cytoplasm. Thus, the splicing reaction can reset the nuclear retention state caused by ESEs, allowing nuclear export of mature mRNAs. Our results reveal a novel aspect of ESE activity that should contribute to gene expression and RNA quality control.

Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus.

Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF(65) were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.

Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme.

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm.

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.