The Function of Pre-mRNA Alternative Splicing in Mammal Spermatogenesis

Abstract

Alternative pre-mRNA splicing plays important roles in co-transcriptional and post-transcriptional regulation of gene expression functioned during many developmental processes, such as spermatogenesis. The studies focusing on alternative splicing on spermatogenesis supported the notion that the development of testis is regulated by a higher level of alternative splicing than other tissues. Here, we aim to review the mechanisms underlying alternative splicing, particularly the splicing variants functioned in the process of spermatogenesis and the male infertility. There are five points regarding the alternative splicing including ⅰ) a brief introduction of alternative pre-mRNA splicing; ⅱ) the alternative splicing events in spermatogenesis-associated genes enriched in different stages of spermatogenesis; ⅲ) the mechanisms of alternative splicing regulation, such as splicing factors and m⁶A demethylation; ⅳ) the splice site recognition and alternative splicing, including the production and degradation of abnormal transcripts caused by gene variations and nonsense-mediated mRNA decay, respectively; ⅴ) abnormal alternative splicing correlated with male infertility. Taking together, this review highlights the impacts of alternative splicing and splicing variants in mammal spermatogenesis and provides new insights of the potential application of the alternative splicing into the therapy of male infertility.

Keywords: Alternative splicing; Male infertility; Mammal spermatogenesis; Splicing factors.

Figure 1

The critical genes and their alternative splicing transcripts in the first wave of spermatogenesis. C-kit is the marker of differentiated spermatogonial cells. Two transcripts, full length transcript and the exon skipped transcript were detected in mouse testis , . Sycp3 and Dmc1 are the markers of spermatocytes. The A3SS transcript and retained intron transcript of Sycp3 were identified in the testes from 60-d and 180-d Large White pigs . The exon skipped Dmc1-d transcript was expressed in both male and female germ cells . Akap4 is the marker of spermatids. Two Akap4 alternative splicing variants - Akap82 and Fsc1 were different in their 5' UTR .

Figure 2

m⁶A modification plays a critical role in mRNA alternative splicing and stability in mammal spermatogenesis. m⁶A methyltransferase Mettl3 is essential for spermatogonial differentiation and meiosis during mouse spermatogenesis. m⁶A modification that mediated by Mettl3 ensures exons containing m⁶A sites have the correct exon inclusion levels . m⁶A demethylase Alkbh5 is required for the late meiotic and spermiogenesis during spermatogenesis . m⁶A tends to mark the 3'-UTRs of longer mRNAs that are destined to be degraded and Alkbh5 controls correct splicing of exon and 3'-UTR which have m⁶A sites , .

Figure 3

The composition of the spliceosome complex and the pre-mRNA splicing process. U1 snRNP binds to the 5' GU. U2 snRNP and SF1 bind to the branch site under the assistance of the mRNA splicing factor U2 associated factor (U2AF65 and U2AF35) and form the splicing complex precursor (A complex) . U4-U5-U6 snRNP trimer forms the splicing complex (B complex) through interactions between RNA-RNA (SR proteins) that bind to ISE and RNA-proteins (hnRNP) that bind to ISS. The 5' end and 3' end of the intron are cleaved from the upstream and downstream exons by a 2', 5'-phosphodiester linkage, and therefore exons are connected together.

Figure 4

Mechanistic model underlying nonsense-mediated mRNA decay. Upf1 interacts with Upf2, Upf3 and then undergoes Smg1-mediated phosphorylation. Pp2a interacts with Smg5, Smg6 and Smg7 and promotes Upf1 de-phosphorylation. Smg5 and Smg6 identify PTC; Smg7 degrade mRNA 5'cap structure and phosphorylated-Upf1 degrades mRNAs -.

Figure 5

The mutations in AR gene splice sites result in aberrant splicing which are closely associated with human androgen insensitivity syndrome. Boxes represent exons of human AR gene. Exon 1 encodes N-terminal transactivation domain (NTD); Exon 2 and 3 encode the first and second zinc fingers of DNA-binding domain (DBD); Exon 4 encodes the hinge region; Exon 5-8 encode the COOH-terminal domain (CTD). Several mutations in AR gene splice sites result in aberrant splicing in androgen insensitivity syndrome patients. For example, the c.1769-1G>A mutation in intron 2 splice acceptor site results in an insertion of 69 nucleotides. The insertion between the two zinc fingers of the AR DNA binding domain (DBD domain) impairs the specific binding of AR response elements to AR , . A c.2449+5G>T mutation in intron 6 boundary splice donor site gives rise to a truncated protein that lacks part of C-terminal ligand-binding domain . A c2667C>T mutation in exon 8 produces an aberrant splicing variant that leads to partial skipping of exon 8 and a shortened 3'-untranslated region and the androgen-induced transcriptional activity is inhibited .

Figure 6

Pre-mRNA splicing in mammal spermatogenesis and male infertility. In normal condition, pre-mRNA undergoes constitutive splicing, removes introns and joins adjacent exons. m⁶A modification maintains correct exon inclusion levels and plays a critical role in mammal spermatogenesis -. Alternative splicing often comes into being along with the emergence of the abnormal splice site recognition. On the one hand, alternative splicing generates abnormal transcripts with a premature termination codon (PTC), which are degraded by nonsense-mediated mRNA decay (NMD) , , . On the other hand, the mutations in splice sites result in the production of exon-skipped transcripts which are closely associated with male infertility , , -.