CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

Abstract

ADP-ribosylation factor collaborator (CARF), which is also known as CDKN2AIP, was first recognized as an ADP-ribosylation factor-interacting protein that participates in the activation of the ARF-p53-p21 (WAF1) signaling pathway under different conditions, such as oxidative and oncogenic stresses. The activation of this pathway often leads to cell growth arrest and apoptosis as well as senescence. Previous studies revealed that CARF, an RNA-binding protein, is critical for maintaining stem cell pluripotency and somatic differentiation. Nevertheless, its involvement in spermatogenesis has not been well examined. In this study, we show that male mice deficient in Carf expression present impaired spermatogenesis and fertility. IP-MS and RNA-seq analyses reveal that CARF/Carf interacts with multiple key splicing factors, such as PABPC1, and directly targets 356 different types of mRNAs in spermatocytes. Carf-associated mRNAs display aberrant splicing patterns when Carf expression is deficient. In addition, our results demonstrate that PIWIL1 expression and localization are altered in the Carf-/- mouse model through the downregulation of PABPC1, which further affects the ratio of pachytene-piRNA. Our study suggests that CARF is critical for regulating alternative splicing in mammalian spermatogenesis and determining infertility in male mice.

1. Introduction

Spermatogenesis refers to the dynamic biological development that takes place in seminiferous tubules [1]. Spermatogonia can self-renew and de-different into spermatogonial stem cell to replenish the pool of stem cells [2]. Type B spermatogonia are generated from type A spermatogonia before they develop into spermatocytes through mitosis and proliferation. Round spermatids, which are the first haploid cells, are subsequently generated after primary spermatocyte division. Finally, round spermatids undergo sperm deformation to form spermatozoa [3,4]. This complicated physiological process is precisely maintained and regulated by several genes at different levels [5–8]. Alternative splicing has been well demonstrated to occur particularly in the testis [9]. However, how this process affects spermatogenesis is not well understood. RNA-binding proteins can not only regulate alternative splicing of mRNAs but also participate in their transportation and translation and play a key role in spermatogenesis [10]. In fact, accumulating evidence suggests that RNA-binding proteins may play a more important role in regulating exon inclusion and protein expression than previously thought [11].

The functions of many RNA-binding proteins have been well studied; for example, poly-A binding protein cytoplasmic 1 (PABPC1) is abundantly expressed in round spermatids before elongation, during which it regulates the expression of an alternative exome that is indispensable for the completion of sperm maturation [12,13]. Spermatogenetic downregulation of RNA-binding proteins, such as PABPC1, is known to be associated with greater befit from retroelements, especially during the elongation of spermatids [14]. Exploring the upstream and downstream proteins of PABPC1 is also the main way to analyze the process of spermatogenesis [15].

Alternatively, in the haploid phase of spermatogenesis, p-element-induced wimpy testis like 1 (PIWIL1) is known to be associated with maintaining normal sperm development in the translational regulation of post-meiotic mRNAs [16]. PIWIL1 binds to PABPC1 and collectively regulates spermatogenesis [17]. If the expression and function of any of these proteins are affected, it can lead to development abnormalities and cause spermatogenesis disorders [18,19].

Many epigenetic mechanisms, including alternative splicing, which is among the most important, have been proposed to regulate transcription activity. After alternative splicing, a single gene can be transcribed into a variety of mRNA and protein isoforms that present different or even contradictory functional and structural characteristics [20]. A previous study indicated that during spermatogenesis, alternative pre-mRNA splicing is critical for the transcriptional regulation of gene expression and function [21]. Recent high-throughput analyses have shown that alternative splicing can occur in more than 95% of all human genes and 60% of mouse genes [22]. Several patterns of alternative splicing events have been identified, such as exon skipping (ES), intron retention (IR), alternative first exon (AFE) as well as mutually exclusive exon (MXE) [23]. Previous studies have shown that aberrant alternative splicing of genes related to reproductive development could result in impaired spermatogenesis and male infertility.

When it was first discovered, CARF was considered an alternative reading frame-interacting protein [24] and could inhibit the transcription of HDM2 as well as P53 signaling pathways [25]. Numerous studies have demonstrated the importance of CARF in spermatogenesis. For example, studies have shown that the number and quality of spermatozoa at 8 weeks of age are sig