RNA-binding Motif Gene Research Articles

RBM-5 modulates U2AF large subunit-dependent alternative splicing in C. elegans

ABSTRACTA key step in pre-mRNA splicing is the recognition of 3ʹ splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor gene RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3ʹ splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.

Expression analysis of RNA-binding motif gene on Y chromosome (RBMY) protein isoforms in testis tissue and a testicular germ cell cancer-derived cell line (NT2).

RNA-binding motif gene on Y chromosome (RBMY), a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility.

Expression of RBMX After Spinal Cord Injury in Rats

RBMX (RNA-binding motif gene, X chromosome) is a heterogeneous nuclear ribonucleoprotein that associates with the spliceosome, binds RNA, and influences alternative splicing. The gene encoding RBMX is located on chromosome Xq26 and is ubiquitously expressed. However, its expression and function in spinal cord injury are still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of RBMX expression in spinal cord. Western blot and immunohistochemistry analysis revealed that RBMX was present in normal spinal cord. It gradually increased, reached a peak at 1 day, and then declined to basal levels at 14 days after spinal cord injury. Double immunofluorescence staining showed that RBMX immunoreactivity was found in neurons and astrocytes, but not in microglia. Interestingly, RBMX expression was increased predominantly in neurons and astrocytes. Moreover, colocalization of RBMX/proliferating cell nuclear antigen (PCNA) and RBMX/active caspase-3 was detected in GFAP and NeuN, respectively. We also examined the expression profiles of active caspase-3, bcl-2, Bax, and PCNA, whose changes were correlated with the expression profiles of RBMX. In conclusion, this is the first description of RBMX expression in spinal cord injury. Our results suggested that RBMX might play crucial roles in CNS pathophysiology after SCI.

Male germ cell-specific RNA binding protein RBMY: a new oncogene explaining male predominance in liver cancer.

Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen-peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome-specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC.

An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains.

The testis‐specific human protein RBMY recognizes RNA through a novel mode of interaction

The RBMY (RNA-binding motif gene on Y chromosome) protein encoded by the human Y chromosome is important for normal sperm development. Although its precise molecular RNA targets are unknown at present, it is suggested that human RBMY (hRBMY) participates in splicing in the testis. Using systematic evolution of ligands by exponential enrichment, we found that RNA stem-loops capped by a C(A)/(U)CAA pentaloop are high-affinity binding targets for hRBMY. Subsequent nuclear magnetic resonance structural determination of the hRBMY RNA recognition motif (RRM) in complex with a high-affinity target showed two distinct modes of RNA recognition. First, the RRM beta-sheet surface binds to the RNA loop in a sequence-specific fashion. Second, the beta2-beta3 loop of the hRBMY inserts into the major groove of the RNA stem. The first binding mode might be conserved in the paralogous protein heterogeneous nuclear RNP G, whereas the second mode of binding is found only in hRBMY. This structural difference could be at the origin of the function of RBMY in spermatogenesis.

Associations of homologous RNA-binding motif gene on the X chromosome (RBMX) and its like sequence on chromosome 9 (RBMXL9) with non-obstructive azoospermia

To investigate the associations of autosomal and X-chromosome homologs of the RNA-binding-motif (RNA-binding-motif on the Y chromosome, RBMY) gene with non-obstructive azoospermia (NOA), as genetic factors for NOA may map to chromosomes other than the Y chromosome. Genomic DNA was extracted using a salting-out procedure after treatment of peripheral blood leukocytes with proteinase K from Japanese patients with NOA (n=67) and normal fertile volunteers (n=105). The DNA were analyzed for RBMX by expressed sequence tag (EST) deletion and for the like sequence on chromosome 9 (RBMXL9) by microsatellite polymorphism. We examined six ESTs in and around RBMX and found a deletion of SHGC31764 in one patient with NOA and a deletion of DXS7491 in one other patient with NOA. No deletions were detected in control subjects. The association study with nine microsatellite markers near RBMXL9 revealed that D9S319 was less prevalent in patients than in control subjects, whereas D9S1853 was detected more frequently in patients than that in control subjects. We provide evidence that deletions in or around RBMX may be involved in NOA. In addition, analyses of markers in the vicinity of RBMXL9 on chromosome 9 suggest the possibility that variants of this gene may be associated with NOA. Although further studies are necessary, this is the first report of the association between RBMX and RBMXL9 with NOA.

Expression of CDY1 may identify complete spermatogenesis

Objective: To investigate the expression of deleted in azoospermia (DAZ), RNA-binding motif (RBM), and chromodomain y1 (CDY1) genes in the testes of men with azoospermia with variable histopathologies. Design: Prospective study. Setting: Andrology laboratory of a university-affiliated maternity hospital. Patient(s): Sixty-six men with azoospermia. Intervention(s): Testicular sperm extraction. Main Outcome Measure(s): The results of gene expression in testicular tissue tested by RT-PCR were correlated with those of histopathologically and microscopically examined minced testicular tissue. Y chromosome microdeletion testing and karyotyping were performed, as was direct sequencing of CDY1-PCR products. Result(s): CDY1-minor expression was detected in all biopsies in which mature spermatids/spermatozoa were observed by histological analysis and/or in the minced tissue. CDY1-minor expression was also detected in two biopsies with arrest at the spermatocyte stage during which no mature spermatids/spermatozoa were observed. A previously unreported CDY1-minor alternative splicing transcript was identified. DAZ and RBM gene expressions were detected in all biopsies in which at least a few germinal cells in early stages were found and in one biopsy histologically determined as Sertoli cell only. Conclusion(s): Our preliminary results suggest that CDY1-minor expression might increase the prospect for complete spermatogenesis, while RBM and DAZ expression can only be indicative of the presence of germinal cells.

Absence of RBM expression as a marker of intratubular (in situ) germ cell neoplasia of the testis

RBM (RNA-binding motif) protein is a marker of male germ cells. This protein is encoded by the Azoospermia factor region-b (AZF-b) of the human Y chromosome and is expressed exclusively in the male germ cell line, that is, spermatogonia, spermatocytes, and round spermatids. The authors analyzed the expression of the RBM gene in germ cell tumors and in the seminiferous tubules in the vicinity of these tumors to identify the presence of IGCN. Sections from testicular germ cell tumors of 21 patients were stained with anti-RBM antibody by using an immunohistochemical method. Distal tubules showing spermatogenesis were immunopositive for RBM protein. All of the germ cell tumors studied were completely immunonegative for RBM. Defined areas of IGCN also showed an absence of RBM expression. Tubules with spermatocyte-like cells, which were expected to express RBM, did not express this protein. This result enabled the identification of tubules as being IGCN. RBM is a novel marker consistently expressed in normal male germ cells but not in malignant germ cell tumors or IGCN. Thus, the absence of RBM expression in germ cells provides a new diagnostic tool of preinvasive malignancy of the testis. H UM P ATHOL 31:1116-1120.

Mammalian Y chromosome evolution and the male-specific functions of Y chromosome-borne genes.

All mammals have an XY chromosomal sex determining system, in which a small Y chromosome triggers male development, and contains genes required for spermatogenesis. The X and Y chromosomes were originally homologous, but diverged during evolution as the Y chromosome was degraded progressively. Comparisons among the sex chromosomes of different mammal groups indicate that the X and Y chromosomes received additions of material from other chromosomes. Genes on the Y chromosome originated from the ancient X-Y pair, or from these additions, or were copies of genes on one of the autosomes. Only genes with important male-specific functions, such as sex determination and spermatogenesis, are selected for and retained on the differential region of the Y chromosome. The mammalian sex determining gene, SRY, controls the testis determination pathway, which includes at least one related gene. Several candidate spermatogenesis genes have been identified, but so far the only one that is conserved on the Y chromosome of all therian mammals is RBM (RNA-binding motif gene, Y chromosome).

Male Infertility and the Y Chromosome

Although it has been established since the 1970s thatdeletions of the long arm of the Y chromosome are as-sociated with spermatogenic failure, only in the last fewyears have these regions been described at the molecularlevel. In parallel, Y-linked genes and gene families thatmay explain the phenotypes of men carrying these de-letions have been identified. The first association be-tween spermatogenic failure and an underlying geneticcause was demonstrated by Tiepolo and Zuffardi (1976)in a report of six azoospermic patients carrying micro-scopically detectable deletions of the distal portion ofYq. In four patients, the deletion was de novo—that is,their fathers were tested and were found to carry intactY chromosomes. On this basis, Tiepolo and Zuffardi(1976) proposed the existence of a spermatogenesis fac-tor, called the “azoospermia factor” (AZF), encoded bya gene on distal Yq. However, the assumption that AZFrepresented a single Y-linked gene was overturned whenVogt et al. (1996) observed that Y chromosome micro-deletions follow a certain deletion pattern, with threerecurrently deleted nonoverlapping subregions in prox-imal, middle, and distal Yq11, designated “AZFa,”“AZFb,” and “AZFc,” respectively. In addition, it be-came clear that these deletions were not exclusively as-sociated with azoospermia (Reijo et al. 1996