Alterations in CDH15 and KIRREL3 in Patients with Mild to Severe Intellectual Disability

Cell-adhesion molecules play critical roles in brain development, as well as maintaining synaptic structure, function, and plasticity. Here we have found the disruption of two genes encoding putative cell-adhesion molecules, CDH15 (cadherin superfamily) and KIRREL3 (immunoglobulin superfamily), by a chromosomal translocation t(11;16) in a female patient with intellectual disability (ID). We screened coding regions of these two genes in a cohort of patients with ID and controls and identified four nonsynonymous CDH15 variants and three nonsynonymous KIRREL3 variants that appear rare and unique to ID. These variations altered highly conserved residues and were absent in more than 600 unrelated patients with ID and 800 control individuals. Furthermore, in vivo expression studies showed that three of the CDH15 variations adversely altered its ability to mediate cell-cell adhesion. We also show that in neuronal cells, human KIRREL3 colocalizes and interacts with the synaptic scaffolding protein, CASK, recently implicated in X-linked brain malformation and ID. Taken together, our data suggest that alterations in CDH15 and KIRREL3, either alone or in combination with other factors, could play a role in phenotypic expression of ID in some patients.     

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.     

Nuclear localization of the pre-mRNA associating protein THOC7 depends upon its direct interaction with Fms tyrosine kinase interacting protein (FMIP)

THOC7 and Fms-interacting protein (FMIP) are members of the THO complex that associate with the mRNA export apparatus. FMIP is a nucleocytoplasmic shuttling protein with a nuclear localization signal (NLS), whereas THOC7 does not contain a typical NLS motif. We show here that THOC7 (50-137, amino acid numbers) binds to the N-terminal portion (1-199) of FMIP directly. FMIP is detected mainly in the nucleus. In the absence of exogenous FMIP, THOC7 resides mainly in the cytoplasm, while in the presence of FMIP, THOC7 is transported into the nucleus with FMIP. Furthermore, THOC7 lacking the FMIP binding site does not co-localize with FMIP, indicating that THOC7/FMIP interaction is required for nuclear localization of THOC7.

Structured summary

MINT-6799962, MINT-6799973, MINT-6800005: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with THOC5 (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800108: FMIP (uniprotkb:Q13769) and THOC7 (uniprotkb: Q6I9Y2) co-localize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800052: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC1 (uniprotkb: Q96FV9) by anti tag coimmunoprecipitation (MI:0007)MINT-6800022: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with FMIP (uniprotkb:Q6DFL5) by pull down (MI:0096)MINT-6799989: THOC7 (uniprotkb:Q6I9Y2) binds (MI:0407) to FMIP (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800071, MINT-6800089: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC7 (uniprotkb:Q6I9Y2) and THOC1 (uniprotkb:Q96FV9) by anti tag coimmunoprecipitation (MI:0007)     

A new view of mRNA export: separating the wheat from the chaff

Current models for the export of messenger RNA share the notion that the highly abundant class of nuclear RNA-binding proteins--the hnRNP proteins--have a key role in exporting RNA. But recent studies have led to a new understanding of several non-hnRNP proteins, including SR proteins and the conserved mRNA export factor ALY, which are recruited to the mRNA during pre-mRNA splicing. These studies, together with older work on hnRNP particles and assembly of the spliceosome, lead us to a new view of mRNA export. In our model, the non-hnRNP factors form a splicing-dependent mRNP complex that specifically targets mature mRNA for export, while hnRNP proteins retain introns in the nucleus. A machinery that is conserved between yeast and higher eukaryotes functions to export the mRNA.     

Identification and characterization of the mouse nuclear export factor (Nxf) family members

TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm.     

Analysis of Nuclear Export Sequence Regions of FUS-Related RNA-Binding Proteins in Essential Tremor

Background and Objective

Genes encoding RNA-binding proteins, including FUS and TDP43, play a central role in different neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Recently, a mutation located in the nuclear export signal (NES) of the FUS gene has been reported to cause an autosomal dominant form of familial Essential tremor.

Material and Methods

We sequenced the exons coding the NES domains of five RNA-binding proteins (TARDBP, hnRNPA2B1, hnRNPA1, TAF15 and EWSR1) that have been previously implicated in neurodegeneration in a series of 257 essential tremor (ET) cases and 376 healthy controls. We genotyped 404 additional ET subjects and 510 healthy controls to assess the frequency of the EWSR1 p.R471C substitution.

Results

We identified a rare EWSR1 p.R471C substitution, which is highly conserved, in a single subject with familial ET. The pathogenicity of this substitution remains equivocal, as DNA samples from relatives were not available and the genotyping of 404 additional ET subjects did not reveal any further carriers. No other variants were observed with significant allele frequency differences compared to controls in the NES coding regions.

Conclusions

The present study demonstrates that the NES domains of RNA-binding proteins are highly conserved. The role of the EWSR1 p.R471C substitution needs to be further evaluated in future studies.     

Both ran and importins have the ability to function as nuclear mRNA export factors

The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.     

CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)–cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.     

The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.     

The mRNA export pathway in plants

A double lipid bilayer separating the nucleus from the cytoplasm, termed the nuclear envelope, is a defining feature of eukaryotes. Nucleocytoplasmic transport of macromolecules through the nuclear pores enables fine-tuned regulation of biologic processes. All mature mRNAs are delivered to the cytoplasm from the nucleus via an mRNA export pathway. Much work has been done in yeast and animals to study the machinery of mRNA export. However, until recently, research on plant mRNA export has been quite limited. Genetic, bioinformatic, and biochemical investigations have expanded our understanding of the mRNA export process in plants. Here, we review recent progress that has been made elucidating the components of the mRNA export pathway in plants. MOS3 (MODIFIER OF SNC1, 3) /AtNup96 and AtNup160 are both components of the highly conserved Nup107–160 nucleoporin complex and were shown to play key roles in mRNA export. MOS11 (MODIFIER OF SNC1, 11), which is homologous to the RNA helicase enhancer CIP29 in human, was recently found to be involved in the same pathway as MOS3. A DEAD Box RNA helicase, LOS4 (low expression of osmotically responsive genes 4) was also found to play a role in the mRNA export process, putatively by carrying mRNA molecules through the nuclear envelope. Recently, a protein complex homologous to the yeast TREX-2 complex was also found to play important roles in mRNA export in plants. It appears that most players in the mRNA export pathway are highly conserved among plants, yeast and animals.     

A Masked PY-NLS in Drosophila TIS11 and Its Mammalian Homolog Tristetraprolin

Many RNA-binding proteins (RBPs) dynamically shuttle between the nucleus and the cytoplasm, often exerting different functions in each compartment. Therefore, the nucleo-cytoplasmic distribution of RBPs has a strong impact on their activity. Here we describe the localization and the shuttling properties of the tandem zinc finger RBP dTIS11, which is the Drosophila homolog of mammalian TIS11 proteins. Drosophila and mammalian TIS11 proteins act as destabilizing factors in ARE-mediated decay. At equilibrium, dTIS11 is concentrated mainly in the cytoplasm. We show that dTIS11 is a nucleo-cytoplasmic shuttling protein whose nuclear export is mediated by the exportin CRM1 through the recognition of a nuclear export signal (NES) located in a different region comparatively to its mammalian homologs. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This NLS partially overlaps the second zinc finger ZnF2. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zinc ion unmask dTIS11 and TTP NLS and promote nuclear import. All together, our results indicate that the nuclear export of TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism may rely on a highly conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.     

Isolation of synthetic lethal mutations in combination with spnab2 of fission yeast

spNab2 is a fission yeast, Schizosaccharomyces pombe, homologue of the budding yeast Nab2 protein that is an essential poly(A)⁺ RNA-binding protein required for both nuclear export of mRNA to cytoplasm and poly(A)⁺ tail length control. Here we performed a synthetic lethal genetic screen in the fission yeast to isolate mutants that are genetically linked to spnab2. We isolated three mutants that showed synthetic lethality under the repressed condition of the spnab2 expression. These mutants defined in different complementation groups. All the mutants exhibited the accumulation of poly(A)⁺ RNA in the nucleus under the restricted condition. In addition, the growth defects of one mutant (SLnab2) were complemented partially by some genes (mlo3 and rae1) required for mRNA export, while those of the rest (SLnab1 and SLnab3) were not complemented by any S. pombe genes we tested, which were known to be involved in mRNA export. These results suggest that the isolated mutants might harbor mutations in novel genes functionally linked to the spnab2 gene.