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.     

家蚕Aly/REF的基因克隆、序列分析及其细胞定位

RNA和输出因子结合蛋白（RNA and export factor binding proteins, REF/Aly）参与RNA的稳定、加工和输出。本研究参照已公开的家蚕Bombyx mori aly序列（GenBank登录号： DQ497195.1）,通过RT-PCR克隆了家蚕aly/ref基因（Bmaly/ref）。测序结果显示,该基因的开放读码框为765 bp,编码254个氨基酸残基, BmAly/REF与黑腹果蝇Drosophila melanogaster、小鼠Mus musculus的同源体的氨基酸序列一致性分别为49.7%和52.7%。结构预测结果显示,BmAly/REF具有REF家族的与RNA结合的结构域RRM,N和C端分别具有REF-N和REF-基序。进化分析结果显示,昆虫的ALY/REF聚为一类,BmAly/REF与赤拟谷盗Tribolium castaneum、意大利蜜蜂Apis mellifera的Aly/REF较为接近。将Bmaly/ref基因克隆进pGS21a(+)载体进行原核表达,重组蛋白免疫小鼠,获得鼠抗BmAly/REF多抗。免疫荧光实验结果显示,BmAly/REF在细胞质和细胞核中均有分布,但主要分布在细胞核中。芯片数据分析显示Bmaly/ref基因在家蚕幼虫5龄第3天各组织中均有较高水平的表达。研究结果显示BmAly/REF可能在RNA的核输出方面发挥作用,为进一步探讨BmAly/REF的功能奠定了基础。     

ICP27 interacts with the RNA export factor Aly/REF to direct herpes simplex virus type 1 intronless mRNAs to the TAP export pathway

Herpes simplex virus type 1 (HSV-1) protein ICP27 facilitates the export of viral intronless mRNAs. ICP27 shuttles between the nucleus and cytoplasm, which has been shown to require a leucine-rich nuclear export sequence (NES). ICP27 export was reported to be sensitive to the CRM1 inhibitor leptomycin B (LMB) in HSV-1-infected cells but not in Xenopus oocytes, where ICP27 interacts with the export factor Aly/REF to access the TAP export pathway. Here, we show that ICP27 interacts with Aly/REF in HSV-1-infected mammalian cells and that Aly/REF stimulates export of viral intronless RNAs but does not cross-link to these RNAs. During infection, Aly/REF was no longer associated with splicing factor SC35 but moved into structures that colocalized with ICP27, suggesting that ICP27 recruits Aly/REF from spliceosomes to viral intronless RNAs. Further, ICP27 was found to interact in vivo with TAP but not with CRM1. In vitro export assays showed that ICP27 export was not sensitive to LMB but was blocked by a dominant-negative TAP deletion mutant lacking the nucleoporin interaction domain. These data suggest that ICP27 uses the TAP pathway to export viral RNAs. Interestingly, the leucine-rich N-terminal sequence was required for efficient export, even though ICP27 export was LMB insensitive. Thus, this region is required for efficient ICP27 export but does not function as a CRM1-dependent NES.     

Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM, and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair.     

Transcription: surprising role for an elusive small nuclear RNA

Since 7SK was discovered as an abundant small nuclear RNA in the mid-70s, its role has remained tantalizingly unsolved. Two recent studies have shown that 7SK acts as a negative regulator of the RNA polymerase II elongation factor P-TEFb, and that this activity is influenced by stress response pathways.     

The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.     

Subnuclear particles containing a small nuclear RNA and heterogeneous nuclear RNA

Five of the stable low molecular weight RNA species in the HeLa cell nucleus have been localized in RNP complexes in the cell nucleus. The two abundant species C and D and the three minor species F, G′ and H are found in RNP particles following two different methods of preparation. Sonication of nuclei releases the five small RNAs and also the hnRNA in RNPs that sediment in a range from 10 to 150 S. Alternatively, incubation of intact nuclei at elevated temperature and pH releases four of the small RNAs and degraded hnRNA in more slowly sedimenting structures.When nuclear RNPs obtained by sonication are digested with RNAase in the presence of EDTA, the hnRNA is degraded and the hnRNPs sediment at 30 S. The structures containing the small RNA species D are similarly shifted to 30 S particles by RNAase and EDTA but not by either agent alone. In contrast, the sedimentation of complexes containing species G′ and H are not altered by exposure to RNAase/EDTA and small RNA species C and F are unstable under these conditions.In isopycnic metrizamide/²H₂O gradients species D and hnRNA accumulate at a density characteristic of RNP particles. They have a similar but not identical distribution.Species D is released from large RNPs by salt concentrations of 0.1 m-NaCl or greater, while the hnRNA remains in large RNP particles. In contrast, the structures containing species G′ and H are stable in 0.3 m-NaCl. All five of the small nuclear RNA species and the hnRNAs are released from rapidly sedimenting complexes by the ionic detergent sodium deoxycholate.It is suggested that the low molecular weight RNA species play a structural role in RNP particles in the cell nucleus and that a subpopulation of species D may be associated with the particles that package the hnRNA.     

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.