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Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. Earlier work has provided indirect evidence of rare somatic mutations in autoreactive T-lymphocytes in multiple sclerosis (MS) patients but such mutations have not been identified thus far. We analysed somatic mutations in blood in 16 patients with relapsing MS and 4 with other neurological autoimmune disease. To facilitate the detection of somatic mutations CD4+, CD8+, CD19+ and CD4-/CD8-/CD19- cell subpopulations were separated. We performed next-generation DNA sequencing targeting 986 immune-related genes. Somatic mutations were called by comparing the sequence data of each cell subpopulation to other subpopulations of the same patient and validated by amplicon sequencing. We found non-synonymous somatic mutations in 12 (60%) patients (10 MS, 1 myasthenia gravis, 1 narcolepsy). There were 27 mutations, all different and mostly novel (67%). They were discovered at subpopulation-wise allelic fractions of 0.2%-4.6% (median 0.95%). Multiple mutations were found in 8 patients. The mutations were enriched in CD8+ cells (85% of mutations). In follow-up after a median time of 2.3years, 96% of the mutations were still detectable. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKFZF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPA1 and STAT3). Whether some of this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for future studies. Show less

The ages of puberty, first sexual intercourse and first birth signify the onset of reproductive ability, behavior and success, respectively. In a genome-wide association study of 125,667 UK Biobank participants, we identify 38 loci associated (P < 5 × 10(-8)) with age at first sexual intercourse. These findings were taken forward in 241,910 men and women from Iceland and 20,187 women from the Women's Genome Health Study. Several of the identified loci also exhibit associations (P < 5 × 10(-8)) with other reproductive and behavioral traits, including age at first birth (variants in or near ESR1 and RBM6-SEMA3F), number of children (CADM2 and ESR1), irritable temperament (MSRA) and risk-taking propensity (CADM2). Mendelian randomization analyses infer causal influences of earlier puberty timing on earlier first sexual intercourse, earlier first birth and lower educational attainment. In turn, likely causal consequences of earlier first sexual intercourse include reproductive, educational, psychiatric and cardiometabolic outcomes. Show less

RBM5, a regulator of alternative splicing of apoptotic genes, and its highly homologous RBM6 and RBM10 are RNA-binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation, and its modulation recapitulates or antagonizes the effects of RBM5, 6, and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation. Show less

To evaluate the prognostic significance of six epigenetic biomarkers (AIM1, CDH1, KIF1A, MT1G, PAK3, and RBM6 promoter hypermethlation) in a homogeneous group of prostate cancer patients, following radical prostatectomy (RP). Biomarker analyses were performed retrospectively on tumors from 95 prostate cancer patients all with a Gleason score of 3 + 4 = 7 and a minimum follow-up period of 8 years. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of six genes in primary prostate tumor tissues. Time to any progression was the primary endpoint and development of metastatic disease and/or death from prostate cancer was a secondary endpoint. The association of clinicopathological and biomolecular risk factors to recurrence was performed using the Log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used. At a median follow-up time of 10 years, 48 patients (50.5%) had evidence of recurrence: Biochemical/PSA relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression, the significant factors were: Older age, HR = 0.95 (95% CI: 0.91, 1.0) (P = 0.03), positive lymph nodes HR = 2.11 (95% CI: 1.05, 4.26) (P = 0.04), and decreased hypermethylation of AIM1 HR = 0.45 (95% CI: 0.2, 1.0) (P = 0.05). Methylation status of AIM1 in the prostate cancer specimen may predict for time to recurrence in Gleason 3 + 4 = 7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort. Show less

RBM5 is one member of a group of structurally related genes that includes RBM6 and RBM10. RBM10 maps to Xp11.23, and one allele is inactivated as a result of X chromosome inactivation. Both RBM5 and RBM6 map to 3p21.3, a tumor suppressor region that experiences loss of heterozygosity in the majority of lung cancers. Overexpression of RBM5, which encodes an RNA-binding protein involved in the regulation of alternative splicing and retards ascites associated tumor growth in immunocompromised mice, a phenomenon that may be related to an associated ability to modulate apoptosis. As part of our quest to gain a better understanding of how the proapoptotic activity of RBM5 might contribute to tumor suppressor function, we reviewed all the literature relating to RBM5 expression, with a focus on lung cancer. On the basis of the existing data, we suggest that-to more thoroughly assess the potential involvement of RBM5 as a lung cancer regulatory protein-more research is required regarding (a) the expression of not only full-length RBM5 but all of the alternate variants associated with the locus, in relation to histologic subtype and smoking history, and (b) the mutation status of various genes within the transforming growth factor-alpha signaling pathway, which may function to either directly or indirectly regulate RBM5 activity in RBM5-retaining lung cancers. Show less

RNA-binding motif (RBM) proteins comprise a large family of RNA-binding proteins whose functions are poorly understood. Since some RBM proteins are candidate alternative splicing factors we examined whether one such member of the family, RBM6, exhibited a pattern of nuclear distribution and targeting consistent with this role. Using antibodies raised against mouse RBM6 to immunostain mammalian cell lines we found that the endogenous protein was both distributed diffusely in the nucleus and concentrated in a small number of nuclear foci that corresponded to splicing speckles/interchromatin granule clusters (IGCs). Tagged RBM6 was also targeted to IGCs, although it accumulated in large bodies confined to the IGC periphery. The basis of this distribution pattern was suggested by the targeting of tagged RBM6 in the giant nuclei (or germinal vesicles (GVs)) of Xenopus oocytes. In spread preparations of GV contents RBM6 was localized both to lampbrush chromosomes and to the surface of many oocyte IGCs, where it was confined to up to 50 discrete patches. Each patch of RBM6 labelling corresponded to a bead-like structure of 0.5-1 microm diameter that assembled de novo on the IGC surface. Assembly of these novel structures depended on the repetitive N-terminal region of RBM6, which acts as a multimerization domain. Without this domain, RBM6 was no longer excluded from the IGC interior but accumulated homogeneously within it. Assembly of IGC-surface structures in mammalian cell lines also depended on the oligomerization domain of RBM6. Oligomerization of RBM6 also had morphological effects on its other major target in GVs, namely the arrays of nascent transcripts visible in lampbrush chromosome transcription units. The presence of oligomerized RBM6 on many lampbrush loops caused them to appear as dense structures with a spiral morphology that appeared quite unlike normal, extended loops. This distribution pattern suggests a new role for RBM6 in the co-transcriptional packaging or processing of most nascent transcripts. Show less

Transcription-induced chimerism, a mechanism involving the transcription and intergenic splicing of two consecutive genes, has recently been estimated to account for approximately 5% of the human transcriptome. Despite this prevalence, the regulation and function of these fused transcripts remains largely uncharacterised. We identified three novel transcription-induced chimeras resulting from the intergenic splicing of a single RNA transcript incorporating the two neighbouring 3p21.3 tumour suppressor locus genes, RBM6 and RBM5, which encode the RNA Binding Motif protein 6 and RNA Binding Motif protein 5, respectively. Each of the three novel chimeric transcripts lacked exons 3, 6, 20 and 21 of RBM6 and exon 1 of RBM5. Differences between the transcripts were associated with the presence or absence of exon 4, exon 5 and a 17 nucleotide (nt) sequence from intron 10 of RBM6. All three chimeric transcripts incorporated the canonical splice sites from both genes (excluding the 17 nt intron 10 insertion). Differential expression was observed in tumour tissue compared to non-tumour tissue, and amongst tumour types. In breast tumour tissue, chimeric expression was associated with elevated levels of RBM6 and RBM5 mRNA, and increased tumour size. No protein expression was detected by in vitro transcription/translation. These results suggest that RBM6 mRNA experiences altered co-transcriptional gene regulation in certain cancers. The results also suggest that RBM6-RBM5 transcription-induced chimerism might be a process that is linked to the tumour-associated increased transcriptional activity of the RBM6 gene. It appears that none of the transcription-induced chimeras generates a protein product; however, the novel alternative splicing, which affects putative functional domains within exons 3, 6 and 11 of RBM6, does suggest that the generation of these chimeric transcripts has functional relevance. Finally, the association of chimeric expression with breast tumour size suggests that RBM6-RBM5 chimeric expression may be a potential tumour differentiation marker. Show less

The aim of this study was to examine the expression of the RBM5 tumor suppressor, in relation to RBM6 and RBM10, to obtain a better understanding of the potential role played by these RBM5-related factors in the regulation of RBM5 tumor-suppressor activity. Paired non-tumor and tumor samples were obtained from 73 breast cancer patients. RNA and protein expression were examined by semi-quantitative reverse transcription-polymerase chain reaction and immunoblot, respectively. Data were analyzed using various statistical methods to test for correlations amongst the RBM5-related factors, and between the factors and various pathological parameters. Most notably, RBM5, RBM10v1, and HER2 protein expression levels were elevated in tumor tissue (P < 0.0001). RBM5 and RBM10v1 protein expression were significantly positively correlated (P < 0.001), as were RBM5 and HER2 protein expression (P < 0.01), in both non-tumor and tumor tissue, whereas RBM10v1 and HER2 protein expression were only marginally correlated, in non-tumor tissue (P < 0.05). Interestingly, RBM5 and RBM10v1 protein expression were both deregulated in relation to RNA expression in tumor tissue. RBM10v2 and RBM6 RNA were highly significantly positively correlated in relation to various factors relating to poor prognosis (P < 0.0001). To our knowledge, this study is the first to examine RBM5 expression at both the RNA and protein level in primary breast tumor tissue, and the first to examine expression of all RBM5-related factors in a comprehensive manner. The results provide a graphic illustration that RBM5-related factors are significantly differentially expressed in breast cancer, and suggest complex inter-related regulatory networks involving alternative splicing, oncogenic expression, and tissue-specific function. Show less

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles. Show less

RBM5 is a known modulator of apoptosis, an RNA binding protein, and a putative tumor suppressor. Originally identified as LUCA-15, and subsequently as H37, it was designated "RBM" (for RNA Binding Motif) due to the presence of two RRM (RNA Recognition Motif) domains within the protein coding sequence. Recently, a number of proteins have been attributed with this same RBM designation, based on the presence of one or more RRM consensus sequences. One such protein, RBM3, was also recently found to have apoptotic modulatory capabilities. The high sequence homology at the amino acid level between RBM5, RBM6, and particularly, RBM10 suggests that they, too, may play an important role in regulating apoptosis. It is the intent of this article to ammalgamate the data on the ten originally identified RBM proteins in order to question the existence of a novel family of RNA binding apoptosis regulators. Show less

In searching for a tumor suppressor gene in the 3p21.3 region, we isolated two genes, RBM5 and RBM6. Sequence analysis indicated that these genes share similarity. RBM5 and-to a lesser extent-RBM6 also have similarity to DXS8237E at Xp11.3-11.23, which maps less than 20 kb upstream of UBE1. A homologue of UBE1, UBE1L, is located at 3p21. 3. FISH analysis showed that the distance between UBE1L and RBM5 in 3p21.3 is about 265 kb. DXS8237E and UBE1 on the X chromosome have the same orientation, whereas on chromosome 3 the orientation of RBM5 and that of RBM6 are opposite to the orientation of UBE1L. Presumably, part of the Xp11.3-11.23 region has duplicated to chromosome 3. Part of this region on chromosome 3 may subsequently have duplicated again within the same chromosomal region. Inversion at some stage of the evolution of the human genome would explain the change in orientation of the genes on chromosome 3 compared with that of the genes on the X chromosome. Show less

DEF-3(g16/NY-LU-12) encodes a novel RNA binding protein isolated by positional cloning from an SCLC homozygous deletion region in 3p21.3 and, in parallel, as a differentially expressed gene during myelopoiesis from FDCPmix-A4 cells. DEF-3(g16/NY-LU-12) is ubiquitously expressed during mouse embryogenesis and in adult organs while human hematopoietic tissues showed differential expression. The mouse and human proteins are highly conserved containing two RNA recognition motifs (RRMs) and other domains associated with RNA binding and protein-protein interactions. A database search identified related proteins in human, rat, C. elegans and S. pombe including the 3p21.3 co-deleted gene, LUCA15. Recombinant proteins containing the RRMs of DEF-3(g16/NY-LU-12) and LUCA15 specifically bound poly(G) RNA homopolymers in vitro. These RRMs also show similarity to those of the Hu protein family. Since anti-Hu RRM domain antibodies are associated with an anti-tumor effect and paraneoplastic encephalomyelitis, we tested sera from Hu syndrome patients with the RRMs of DEF-3(g16/NY-LU-12) and LUCA15. These were non-reactive. Thus, DEF-3(g16/NY-LU-12) and LUCA15 represent members of a novel family of RNA binding proteins with similar expression patterns and in vitro RNA binding characteristics. They are co-deleted in some lung cancers and immunologically distinct from the Hu proteins. Show less

In the search for a tumour suppressor gene in the 3p21.3 region we isolated two genes, RBM5 and RBM6. Gene RBM5 maps to the region which is homozygously deleted in the small cell lung cancer cell line GLC20; RBM6 crosses the telomeric breakpoint of this deletion. Sequence comparison revealed that at the amino acid level both genes show 30% identity. They contain two zinc finger motifs, a bipartite nuclear signal and two RNA binding motifs, suggesting that the proteins for which RBM5 and RBM6 are coding have a DNA/RNA binding function and are located in the nucleus. Northern and Southern analysis did not reveal any abnormalities. By SSCP analysis of 16 lung cancer cell lines we found only in RBM5 a single presumably neutral mutation. By RT-PCR we demonstrated the existence of two alternative splice variants of RBM6, one including and one excluding exon 5, in both normal lung tissue and lung cancer cell lines. Exclusion of exon 5 results in a frameshift which would cause a truncated protein of 520 amino acids instead of 1123 amino acids. In normal lung tissue, the relative amount of the shorter transcript was much greater than that in the lung tumour cell lines, which raises the question whether some tumour suppressor function may be attributed to the derived shorter protein. Show less