RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.This work was supported by grants from the National Health and Medical Research Council (NHMRC) to DJ (#606503); the Australian Research Council (ARC) to MKO and CJO; the New South Wales Cancer Council, Cancer Institute New South Wales, Banque Nationale de Paris-Paribas Australia and New Zealand, RT Hall Trust, and the National Breast Cancer Foundation to CJO. DJ was an NHMRC Peter Doherty Postdoctoral Fellow (#384297). MKO and CJO are NHMRC Senior Research Fellows (#545805, #481310). CCG is an NHMRC Australia Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Manual therapy with or without physical medicine modalities for neck pain: a systematic review

Manual therapy interventions are often used with or without physical medicine modalities to treat neck pain. This review assessed the effect of 1) manipulation and mobilisation, 2) manipulation, mobilisation and soft tissue work, and 3) manual therapy with physical medicine modalities on pain, function, patient satisfaction, quality of life (QoL), and global perceived effect (GPE) in adults with neck pain. A computerised search for randomised trials was performed up to July 2009. Two or more authors independently selected studies, abstracted data, and assessed methodological quality. Pooled relative risk (RR) and standardised mean differences (SMD) were calculated when possible. We included 19 trials, 37% of which had a low risk of bias. Moderate quality evidence (1 trial, 221 participants) suggested mobilisation, manipulation and soft tissue techniques decrease pain and improved satisfaction when compared to short wave diathermy, and that this treatment combination paired with advice and exercise produces greater improvements in GPE and satisfaction than advice and exercise alone for acute neck pain. Low quality evidence suggests a clinically important benefit favouring mobilisation and manipulation in pain relief [1 meta-analysis, 112 participants: SMD -0.34(95% CI: -0.71, 0.03), improved function and GPE (1 trial, 94 participants) for participants with chronic cervicogenic headache when compared to a control at intermediate and long term follow-up; but no difference when used with various physical medicine modalities. (C) 2010 Elsevier Ltd. All rights reserve

Manual therapy and exercise for neck pain: A systematic review

Manual therapy is often used with exercise to treat neck pain. This cervical overview group systematic review update assesses if manual therapy, including manipulation or mobilisation, combined with exercise improves pain, function/disability, quality of life, global perceived effect, and patient satisfaction for adults with neck pain with or without cervicogenic headache or radiculopathy. Computerized searches were performed to July 2009. Two or more authors independently selected studies, abstracted data, and assessed methodological quality. Pooled relative risk (pRR) and standardized mean differences (pSMD) were calculated. Of 17 randomized controlled trials included, 29% had a low risk of bias. Low quality evidence suggests clinically important long-term improvements in pain (pSMD-0.87(95% CI: -1.69, -0.06)), function/disability, and global perceived effect when manual therapy and exercise are compared to no treatment. High quality evidence suggests greater short-term pain relief [pSMD-0.50 (95% CI: -0.76,-0.24)] than exercise alone, but no long-term differences across multiple outcomes for (sub)acute/chronic neck pain with or without cervicogenic headache. Moderate quality evidence supports this treatment combination for pain reduction and improved quality of life over manual therapy alone for chronic neck pain; and suggests greater short-term pain reduction when compared to traditional care for acute whiplash. Evidence regarding radiculopathy was sparse. Specific research recommendations are made. (C) 2010 Elsevier Ltd. All rights reserve

Manipulation or mobilisation for neck pain: A Cochrane Review

Manipulation and mobilisation are often used, either alone or combined with other treatment approaches, to treat neck pain. This review assesses if manipulation or mobilisation improves pain, function/disability, patient satisfaction, quality of life (QoL), and global perceived effect (GPE) in adults experiencing neck pain with or without cervicogenic headache or radicular findings. A computerised search was performed in July 2009. Randomised trials investigating manipulation or mobilisation for neck pain were included. Two or more authors independently selected studies, abstracted data, and assessed methodological quality. Pooled relative risk (pRR) and standardised mean differences (pSMD) were calculated. 33% of 27 trials had a low risk of bias. Moderate quality evidence showed cervical manipulation and mobilisation produced similar effects on pain, function and patient satisfaction at intermediate-term follow-up. Low quality evidence suggested cervical manipulation may provide greater short-term pain relief than a control (pSMD -0.90 (95%CI: -1.78 to -0.02)). Low quality evidence also supported thoracic manipulation for pain reduction (NNT 7; 46.6% treatment advantage) and increased function (NNT 5; 40.6% treatment advantage) in acute pain and immediate pain reduction in chronic neck pain (NNT 5; 29% treatment advantage). Optimal technique and dose need to be determined. (C) 2010 Elsevier Ltd. All rights reserve

RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing

RBM5 RRM2 is required for appropriate pre-mRNA splicing.

<p>(<b>A</b>) Relative abundance of <i>St5</i>, <i>Asb1</i> and <i>Plg2g10</i> is significantly increased in the RBM5 pulled down (RBM5-PD) samples compared to that of IgG-PD controls. Error bars = S.D. (standard deviation, <i>n</i> = 3 sets of each pull down). * indicates statistical significance (p&lt;0.05, t-test). (<b>B–D</b>) The R263P mutation in the RRM2 of RBM5 leads to aberrant splicing of <i>St5</i>, <i>Asb1</i> and <i>Pla2g10</i>. Black bars represent exons and open bars represent UTRs. FL: full-length, ex: exon, Δ ex 3 refers to exon 3 skipping, Δ ex 3+ex 4 refers to exon 3 and exon 4 skipping, and + intron refers to intron retention. (<b>E</b>) Splicing defects of <i>Kif17</i>, <i>Anks3</i>, <i>Rangap1</i>, <i>Nfx1</i> and <i>Cftr</i>. Transcripts with obvious shift in their relative intensities are indicated by arrows.</p

Functional clustering of differentially expressed genes identified by microarray analysis of RNAs isolated from round spermatids of <i>Rbm5^sda/sda</i> and <i>Rbm5^WT/WT</i> postnatal day 28 testes.

a<p>Differentially expressed genes associated with a particular significant function are given.</p

ST5 negatively regulates ERK signalling in round spermatids.

<p>(A) Schematic of the mouse ST5 protein. (B) Reduction of the ST5 protein leads to hyperactivation of ERK1/2 in round spermatids. HPRT was used a loading control. (C) ST5 localises strongly in the cytoplasm of round spermatids as determined by immunohistochemistry of postnatal day 28 testis section. (C, insert) No positive signal was observed when the ST5 antibody was omitted. Scale bars = 50 µM.</p

RBM5 target mRNAs in round spermatids identified using RNA pull down coupled with microarrays.

<p>RBM5 target mRNAs in round spermatids identified using RNA pull down coupled with microarrays.</p