SUN5, a testis-specific nuclear membrane protein, participates in recruitment and export of nuclear mRNA in spermatogenesis

Abstract

SUN5, a testis-specific gene, is associated with acephalic spermatozoa syndrome (ASS). Here, we demonstrate that SUN5 is involved in mRNA export. In Sun5-knockout mice (Sun5–/–), poly(A)+ RNA accumulates in the nuclei of germ cells, leading to reduced sperm counts, decreased sperm motility and disrupted sperm head-to-tail junctions. Additionally, in the GC-2 germ cell line with RNA interference of Sun5, heterogeneous nuclear ribonucleoproteins (hnRNPs) and poly (A)+ RNA (mainly mRNA) are retained in the nucleus. Further mechanistic studies reveal that SUN5 interacts with Nxf1 (nuclear RNA export factor 1) and nucleoporin 93 (Nup93). Interference with Nup93 inhibits mRNA export. Treatment with leptomycin B to block the CRM1 pathway indicates that Sun5 regulates mRNA export through an Nxf1-dependent pathway. In Sun5–/– mice, the binding of Nxf1 and Nup93 decreases due to loss of Sun5 function, and the process of submitting Nxf1-binding mRNPs to Nup93 is inhibited, resulting in abnormal spermatogenesis. Together, these data may elucidate a novel pathway for mRNA export in male germ cells.

1. Introduction

Spermatogenesis is a complex process in which spermatogonial stem cells undergo a series of differentiations to produce haploid spermatids, after which the spermatids develop into mature spermatozoa [1]. At each stage, testis-specific genes are involved and exert their specific functions. Genes are transcribed in the nucleus, encased in messenger ribonucleoprotein particles (mRNPs) and translocated to the cytoplasm via the nuclear pore complex (NPC) for immediate translation or storage [2,3]. However, the function and molecular mechanism of these testis-specific genes in mRNA nuclear export are still unclear.

Pre-mRNAs are packaged into mRNPs by loading with different RNA-binding proteins (RBPs). Current studies have demonstrated that in eukaryotes, certain RBPs are deposited at specific locations on pre-mRNAs when transcribed for synthesis [4]. As a large class of RBPs, heterogeneous nuclear ribonucleoproteins (hnRNPs) affect multiple aspects of mRNA metabolism, including the encasing of nascent transcripts, alternative splicing, nucleus-to-cytoplasm transport and translational regulation [5]. In humans, approximately 30 different hnRNPs (called A1 to U) bind to different transcripts synthesized by RNA-pol-II in the nucleus [6]. HnRNPs, such as hnRNP K, depart from mRNPs before being exported or rapidly shuttling back from the cytoplasm to the nucleus [7]. HnRNPs assist in the control of pre-mRNA maturation into mRNAs and accompany the mRNA at different stages [6].

RNA is transported from the nucleus to the cytoplasm in different ways and is broadly categorized into two forms: the chromosomal region maintenance 1 (CRM1) pathway and the nuclear RNA export factor 1 (NXF1) pathway, depending on the transporter receptor [8]. A small subset of mRNAs and various miscellaneous RNAs, such as miRNAs and lncRNAs, are mainly transported by the CRM1 protein, similar to karyopherin [9]. However, the transport of most constitutively expressed mRNAs is mediated by the NXF1-NXT1 heterodimer, despite the variety of mRNAs in terms of splicing, maturation state, size, copy number, etc. [10]. NXF1 consists of four domains: a leucine-rich region (LRR), an RNA recognition motif (RRM), an NTF2-like domain (NTF2L) which binds NXT1 to form the NXF1/NXT1 heterodimer, and a ubiquitin-associated (UBA) domain [11]. In the process of mRNA export, the NXF1-NXT1 heterodimer is loaded onto mRNPs via the transcription export complex (TREX), and mRNPs with export capability are assembled in preparation for translocation [12,13].