Major Spliceosome Defects Cause Male Infertility and Are Associated with Nonobstructive Azoospermia in Humans
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Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans Hao Wua,b,1, Liwei Sunc,d,1, Yang Wena,e,1, Yujuan Liua,b, Jun Yua,b, Feiyu Maoc,f, Ya Wanga,b, Chao Tongg, Xuejiang Guoa,b, Zhibin Hua,e, Jiahao Shaa,b, Mingxi Liua,b,2, and Laixin Xiac,2 aState Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, People’s Republic of China; bDepartment of Histology and Embryology, Nanjing Medical University, Nanjing 210029, People’s Republic of China; cKey Laboratory of Zebrafish Modeling and Drug Screening for Human Diseases of Guangdong Higher Education Institutes, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, People’s Republic of China; dState Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China; eDepartment of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 211166, People’s Republic of China; fState Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China; and gLife Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou 310058, People’s Republic of China Edited by Margaret T. Fuller, Stanford University School of Medicine, Stanford, CA, and approved February 19, 2016 (received for review July 12, 2015) Processing of pre-mRNA into mRNA is an important regulatory Drosophila (9). Therefore, Drosophila provides a simple system mechanism in eukaryotes that is mediated by the spliceosome, a for investigating the complex genetic basis and related mo- huge and dynamic ribonucleoprotein complex. Splicing defects are lecular mechanisms of human male fertility. We recently suc- implicated in a spectrum of human disease, but the underlying cessfully used Drosophila to test the reproductive function of mechanistic links remain largely unresolved. Using a genome-wide conserved genes identified by a genome-wide association study association approach, we have recently identified single nucleotide (GWAS), leading to the identification of seven previously un- polymorphisms in humans that associate with nonobstructive identified factors essential for male fertility (10). azoospermia (NOA), a common cause of male infertility. Here, The vast majority of genes in higher eukaryotic genomes are using genetic manipulation of corresponding candidate loci in Dro- interrupted by introns, which are removed from pre-mRNAs by sophila, we show that the spliceosome component SNRPA1/U2A is the spliceosome. Most pre-mRNAs are processed through the essential for male fertility. Loss of U2A in germ cells of the Dro- major spliceosome pathway, which relies on the major spliceo- sophila testis does not affect germline stem cells, but does result in some, a conserved, very large complex consisting of five small the accumulation of mitotic spermatogonia that fail to differenti- nuclear ribonucleoprotein (snRNP) complexes (U1, U2, U4, U5, ate into spermatocytes and mature sperm. Lack of U2A causes in- and U6) and approximately 150 proteins (11). Compared with sufficient splicing of mRNAs required for the transition of germ the large number of cis-acting splicing mutations discovered in cells from proliferation to differentiation. We show that germ cell- human disease, only a few mutations in spliceosome-associated genes have been shown to cause diseases, including retinal de- specific disruption of other components of the major spliceosome – manifests with the same phenotype, demonstrating that mRNA generation and Taybi Linder syndrome (12). Whether the spli- processing is required for the differentiation of spermatogonia. This ceosome core components are involved in human reproductive requirement is conserved, and expression of human SNRPA1 fully disease remains unknown. Drosophila restores spermatogenesis in U2A mutant flies. We further report In the present study, using as a model, we evalu- ated the function of an SNP locus strongly associated with NOA that several missense mutations in human SNRPA1 that inhibit the assembly of the major spliceosome dominantly disrupt spermatogo- nial differentiation in Drosophila. Collectively, our findings uncover Significance a conserved and specific requirement for the major spliceosome during the transition from spermatogonial proliferation to differ- The major spliceosome is required for mRNA processing and is entiation in the male testis, suggesting that spliceosome defects believed to be essential for cell survival. Here we report that affecting the differentiation of human spermatogonia contribute mutations affecting the major spliceosome are associated with to NOA. nonobstructive azoospermia (NOA), a common but poorly un- derstood cause of male infertility in humans. Specifically, we GWAS | NOA | spliceosome | spermatogonia | spermatogenesis report that spliceosome dysregulation impairs the differentia- tion of spermatogonia, abolishing the maturation of germ cells pproximately 30–55% of cases of infertility in humans are into sperm. The requirement for the major spliceosome during Arelated to male factors, and of these, 10–15% have been spermatogonial differentiation is highly conserved among spe- attributed to azoospermia, defined as the complete absence of cies. Our study has uncovered genetic causes and molecular sperm in semen (1). Nonobstructive azoospermia (NOA), in which mechanisms underlying NOA; these results will likely provide direction for the genetic testing and treatment of patients the testicles fail to produce mature sperm in the ejaculate because with NOA. of abnormal spermatogenesis, accounts for approximately one- half of azoospermia cases (2, 3). NOA is a heterogeneous disorder Author contributions: H.W., C.T., X.G., Z.H., J.S., M.L., and L.X. designed research; H.W., caused by complex genetic and environmental factors. Hereditary L.S., Y. Wen, Y.L., J.Y., F.M., and Y. Wang performed research; H.W. and L.X. analyzed changes, including chromosome abnormalities and gene muta- data; and M.L. and L.X. wrote the paper. tions, have been implicated in NOA (4–6); however, the etiology The authors declare no conflict of interest. of this disease remains largely unclear. This article is a PNAS Direct Submission. Spermatogenesis is one of the most conserved biological pro- 1H.W., L.S., and Y. Wen contributed equally to this work. cesses from Drosophila to humans (7, 8). For example, mutations 2To whom correspondence may be addressed. Email: [email protected] or xlxsq@ in boule cause sterility in Drosophila, and mutants of the human 126.com. homologous gene, DAZ, are associated with azoospermia. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Moreover, DAZ can restore the meiosis of the boule mutant in 1073/pnas.1513682113/-/DCSupplemental. 4134–4139 | PNAS | April 12, 2016 | vol. 113 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1513682113 Downloaded by guest on September 24, 2021 spermatogenesis (Fig. 1B) as a system to test the reproductive function of these genes by performing ubiquitous knockdown of each of the four candidate genes in adult flies using RNA in- terference (RNAi) (15). To bypass the potential lethality of these knockdowns, we used a temperature-sensitive conditional approach incorporating the GAL4 upstream activation sequence (UAS) bipartite expression system with a temperature-sensitive mutant of GAL80 (Gal80[ts]) that reversibly suppresses GAL4 activity at 18 °C but allows GAL4 binding to UAS at 29 °C. In flies transgenic for the three components of this system (UAS-RNAi; GAL-4 driver, here a ubiquitous tub-gal4; and tub-Gal80[ts]), expression of RNAi and resulting knockdown of the target gene were suppressed at permissive temperature, such that tub-gal80[ts]; tub-gal4 > UAS- RNAi males raised at 18 °C were fertile (SI Appendix, Fig. S1C). Knockdown of each of the four target genes on exposure to the restrictive temperature (29 °C) resulted in reduced male fertility (SI Appendix, Fig. S1D). In particular, knockdown of U2A, the homologous gene of hSNRPA1 that is in close proximity to rs7166401, caused complete male sterility (Table 1 and SI Appendix, Fig. S1E). These results suggest the locus 15q26.3 (rs7166401) and hSNRPA1 as a susceptibility locus and a candidate gene, re- spectively, for male infertility. Drosophila Spermatogenesis Requires Availability of U2A in Germ Cells. To validate the outcomes with RNAi-mediated knock- down of U2A, we generated a U2A null mutant fly line using CRISPR/Cas9-mediated genome editing. The resulting null al- GENETICS lele contained a 7-bp deletion in exon 1 (bases 253–259 following Fig. 1. The locus 15q26.3 (rs7166401) is susceptible to male infertility. the U2A start codon), which caused a frame shift and loss of the (A) Regional association plot of SNP rs7166401. SNP rs716640 was identified conserved leucine-rich repeat C-terminal (LRRCT) and U2A by genome-wide association study (GWAS) in Han Chinese men. SNPs are SI Appendix A U2A1 2 domains ( , Fig. S2 ). Flies homozygous for the shown in purple, and r values for other SNPs are indicated in color. Genes allele were not viable. To rescue these flies, we introduced a within the region of interest (1 Mb) are annotated with direction of tran- compensatory transgene (hs-U2A) expressing U2A cDNA under scription marked by arrows. (B) Schematic diagram of spermatogenesis