Structure of the C-terminal FG-nucleoporin binding domain of Tap/NXF1

The vertebrate Tap protein is a member of the NXF family of shuttling transport receptors for nuclear export of mRNA. Tap has a modular structure, and its most C-terminal domain is important for binding to FG repeat-containing nuclear pore proteins (FG-nucleoporins) and is sufficient to mediate nuclear shuttling. We report the solution structure of this C-terminal domain, which is based on a distinctive arrangement of four alpha-helices and is joined to the next module by a flexible 12-residue Pro-rich linker. F617A Tap suppresses FG-nucleoporin binding by the most C-terminal domain that, together with the structure of the other modules from which Tap is constructed, provides a structural context for its nuclear shuttling function.     

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.     

Transcriptome-wide modulation of splicing by the exon junction complex

Background

The exon junction complex (EJC) is a dynamic multi-protein complex deposited onto nuclear spliced mRNAs upstream of exon-exon junctions. The four core proteins, eIF4A3, Magoh, Y14 and MLN51, are stably bound to mRNAs during their lifecycle, serving as a binding platform for other nuclear and cytoplasmic proteins. Recent evidence has shown that the EJC is involved in the splicing regulation of some specific events in both Drosophila and mammalian cells.

Results

Here, we show that knockdown of EJC core proteins causes widespread alternative splicing changes in mammalian cells. These splicing changes are specific to EJC core proteins, as knockdown of eIF4A3, Y14 and MLN51 shows similar splicing changes, and are different from knockdown of other splicing factors. The splicing changes can be rescued by a siRNA-resistant form of eIF4A3, indicating an involvement of EJC core proteins in regulating alternative splicing. Finally, we find that the splicing changes are linked with RNA polymerase II elongation rates.

Conclusion

Taken together, this study reveals that the coupling between EJC proteins and splicing is broader than previously suspected, and that a possible link exists between mRNP assembly and splice site recognition.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0551-7) contains supplementary material, which is available to authorized users.     

Holliday junction resolution: Regulation in space and time

Holliday junctions (HJs) can be formed between sister chromatids or homologous chromosomes during the recombinational repair of DNA lesions. A variety of pathways act upon HJs to remove them from DNA, in events that are critical for appropriate chromosome segregation. Despite the identification and characterization of multiple enzymes involved in HJ processing, the cellular mechanisms that regulate and implement pathway usage have only just started to be delineated. A conserved network of core cell-cycle kinases and phosphatases modulate HJ metabolism by exerting spatial and temporal control over the activities of two structure-selective nucleases: yeast Mus81-Mms4 (human MUS81-EME1) and Yen1 (human GEN1). These regulatory cycles operate to establish the sequential activation of HJ processing enzymes, implementing a hierarchy in pathway usage that ensure the elimination of chromosomal interactions which would otherwise interfere with chromosome segregation. Mus81-Mms4/EME1 and Yen1/GEN1 emerge to define a special class of enzymes, evolved to satisfy the cellular need of safeguarding the completion of DNA repair when on the verge of chromosome segregation.     

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.     

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.     

RanBP2/Nup358 provides a major binding site for NXF1-p15 dimers at the nuclear pore complex and functions in nuclear mRNA export

Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.     

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.     

Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1

Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.     

Arginine methylation of REF/ALY promotes efficient handover of mRNA to TAP/NXF1

The REF/ALY mRNA export adaptor binds TAP/NXF1 via an arginine-rich region, which overlaps with its RNA-binding domain. When TAP binds a REF:RNA complex, it triggers transfer of the RNA from REF to TAP. Here, we have examined the effects of arginine methylation on the activities of the REF protein in mRNA export. We have mapped the arginine methylation sites of REF using mass spectrometry and find that several arginines within the TAP and RNA binding domains are methylated in vivo. However, arginine methylation has no effect on the REF:TAP interaction. Instead, arginine methylation reduces the RNA-binding activity of REF in vitro and in vivo. The reduced RNA-binding activity of REF in its methylated state is essential for efficient displacement of RNA from REF by TAP in vivo. Therefore, arginine methylation fine-tunes the RNA-binding activity of REF such that the RNA–protein interaction can be readily disrupted by export factors further down the pathway.     

Nonsense-mediated translational repression involves exon junction complex downstream of premature translation termination codon

Human transforming growth factor-β receptor type 2 (TGFβR2) mRNA harboring a premature translation termination codon (PTC) generated by frameshift mutation is targeted for nonsense-mediated translational repression (NMTR), rather than nonsense-mediated mRNA decay (NMD). Here we show that exon junction complex (EJC) downstream of a PTC plays an inhibitory role in translation of TGFβR2 mRNA. Translational repression by core EJC components occurs after formation of 80S ribosome complex, which is demonstrated using different types of internal ribosome entry sites (IRESes). Our findings implicate EJCs or core EJC components as negative regulators of translation.     

The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function.

The centromere and promoter factor Cpf1 binds centromere DNA element I found in all centromere DNAs from the yeast Saccharomyces cerevisiae. We analyzed thirty different point mutations in or around CEN6-CDEI (ATCACGTG) for their relative binding affinity to Cpf1 and these data were compared with the in vivo centromere function of these mutants. We show that the minimal length of the Cpf1 binding site needed for full in vitro binding and in vivo activity is 10 base pairs long comprised of CDEI plus the two base pairs 3' of this sequence. The palindromic core sequence CACGTG is most important for in vivo CEN function and in vitro Cpf1 binding. Symmetrical mutations in either halfsite of the core sequence affect in vitro Cpf1 binding and in vivo mitotic centromere function asymmetrically albeit to a different extent. Enlarging the CDEI palindrome to 12 or 20 bps increases in vitro Cpf1 binding but results in increased chromosome loss rates suggesting a need for asymmetrical Cpf1 binding sequences. Additionally, the ability of Cpf1 protein to bind a mutant CDEI element in vitro does not parallel the ability of that mutant to confer in vivo CEN activity. Our data indicate that the in vitro binding characteristics of Cpf1 to CDEI only partly overlap with their corresponding activity within the centromere complex, thus suggesting that in the in vivo situation the CDEI/Cpf1 complex might undergo interactions with other centromere DNA/protein complexes.     

Mammalian UPF3A and UPF3B can activate nonsense‐mediated mRNA decay independently of their exon junction complex binding

Nonsense‐mediated mRNA decay (NMD) is governed by the three conserved factors—UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B‐dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B‐dependent and ‐independent manner. While UPF3A is almost dispensable for NMD in wild‐type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC‐binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved “mid” domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.     

The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex

The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin-based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1-Rac signaling. Tiam1 interacts with Par3 and aPKCzeta, which are two components of the conserved Par3-Par6-aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCzeta, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3-Par6-aPKC polarity complex.     

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.     

TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5'-GA-3' and 5'-UG-3' repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.