Control of messenger RNA fate by RNA-binding proteins: an emphasis on mammalian spermatogenesis

Abstract

Posttranscriptional status of messenger RNAs (mRNA) can be affected by many factors, most of which are RNA-binding proteins (RBP) that either bind mRNA in a nonspecific manner or through specific motifs, usually located in the 3' untranslated regions. RBPs can also be recruited by small noncoding RNAs (sncRNA), which have been shown to be involved in posttranscriptional regulations and transposon repression (eg, microRNAs or P-element-induced wimpy testis-interacting RNA) as components of the sncRNA effector complex. Non-sncRNA-binding RBPs have much more diverse effects on their target mRNAs. Some can cause degradation of their target transcripts and/or repression of translation, whereas others can stabilize and/or activate translation. The splicing and exportation of transcripts from the nucleus to the cytoplasm are often mediated by sequence-specific RBPs. The mechanisms by which RBPs regulate mRNA transcripts involve manipulating the 3' poly(A) tail, targeting the transcript to polysomes or to other ribonuclear protein particles, recruiting regulatory proteins, or competing with other RBPs. Here, we briefly review the known mechanisms of posttranscriptional regulation mediated by RBPs, with an emphasis on how these mechanisms might control spermatogenesis in general.

Figure 1

Delayed translation in late spermiogenesis. Spermatogenesis is a process through which spermatogonial stem cells differentiate into spermatocytes, which then undergo meiotic cell divisions and become round spermatids. Round spermatids undergo morphogenesis, a process termed “spermiogenesis,” and differentiate into spermatozoa, which are the male gamete. During this complex differentiation process, transcription completely ceases upon nuclear condensation and elongation in mid spermiogenesis. Therefore, proteins required for the rest of the differentiation program must be produced through translation using the messenger RNAs synthesized and stored prior to the cease of transcription. As indicates single type A spermatogonia; Ap, paired type A spermatogonia; Aal, aligned type A spermatogonia.

Figure 2

Known mechanisms of posttranscriptional regulation of messenger RNAs (mRNA). (A) Splicing. The top RNA is heterogeneous nuclear RNA (hnRNA) that includes recognition motifs in its exons and introns. Shown in red are enhancers, whereas those shown in green are silencers. Proteins that bind to these sequences are heterogeneous nuclear ribonucleoproteins (hnRNP). Those that encourage exon inclusion are activators, whereas those that discourage exon inclusion are repressors. The mechanism by which they perform this function is by interactions with small nuclear ribonucleoprotein particles (snRNP), particularly U1 and U2. Some hnRNPs remain bound to the transcript after splicing, and will often mediate export from the nucleus. (B) Formation of mature mRNA. Precursor mRNA transcribed from DNA contains motifs that hnRNPs recognize and with which they associate. Splicing occurs simultaneously with cap and tail modifications. This process yields mature mRNA that is exported to the cytoplasm. (C) Transport complex. RNA-binding proteins (RBP) can associate with a STAUFEN complex that associates with kinesin or dynein. This will allow transcripts to be transported along the cytoskeleton to where they are needed. Usually RBPs block the association of the initiation complex, keeping the transcript translationally inactive during transportation. (D) “Closed loop” model of translation initiation. A long poly(A) tail attracts poly(A)-binding protein (PABP), which associates with eukaryotic translation initiation factor 4G (EIF4G). EIF4E has a high affinity for the 59 cap and for EIF4G. An initiation factor complex associates with EIF4G and EIF4E. This complex binds the 40S subunit. The 40S subunit along with several initiation factors (together termed “43S”) will scan the transcript for the start codon, at which point the initiation translational RNA will bind and recruit the large subunit (60S subunit), starting translation.

Figure 3

Regulation of cytoplasmic polyadenylation element– binding protein (CPEB) function by phosphorylation. (A) When not phosphorylated, CPEB will hold the transcript inactive by association with Maskin. Maskin binds EIF4E competitively inhibiting binding by eukaryotic translation initiation factor 4G (EIF4G). This activity is encouraged by the maintenance of a short poly(A) tail. (B) Once phosphorylated, CPEB binds to cleavage and polyadenylation–specific factor (CPSF) that associates with polyadenylate polymerase (PAP). This lengthens the poly(A) tail, causing poly(A)-binding protein (PABP) binding. This encourages association with EIF4G. This occurs in conjunction with a loss of affinity of Maskin for EIF4E, although Maskin remains bound to CPEB. Together, this causes the initiation complex to form and encourages the small ribosomal subunit to bind, which is a limiting step for translation initiation.

Figure 4

Male germinal granules (intermitochondrial cement/nuage and chromatoid bodies) highlighted by immunofluorescent staining of mouse VASA homolog (MVH [DDX4]) and murine PIWI homolog 1 (MIWI [PIWIL1]) in adult mouse testes. Intermitochondrial cement (arrow) is widely distributed in the cytoplasm of all spermatogenic cells, including spermatogonia (Sg), spermatocytes (Sp), and spermatids (Sd), whereas chromatoid bodies (arrowheads) resemble perinuclear bodies mainly found in round spermatids (rSd). MVH (red fluorescence) is associated with male germinal granules in all types of developing germ cells, including intermitochondrial cement in all spermatogenic cells and chromatoid bodies in round spermatids. MIWI (green fluorescence) is predominantly expressed in intermitochondrial cement in pachytene spermatocytes and chromatoid bodies in round spermatids. eSd, elongated spermatids. Purified human anti-MVH/DDX4 antibody (R11-A3 Fab) was purchased from BD Pharmingen Inc (San Diego, California), and affinity-purified rabbit anti-PIWIL1 polyclonal antibodies were prepared by GenScript Corporation (Piscataway, New Jersey). Both antibodies were used at a dilution of 1:100.

Figure 5

Biogenesis of microRNAs (miRNA) and PIWI-interacting RNAs (piRNA). DNA is transcribed into RNA by RNA polymerase II/III, and the resulting primary transcript (pri-miRNA) contains a hairpin structure that is recognized and cleaved by a microprocessor complex consisting of DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5), DGRC8, and DROSHA. This results in a ~70-nucleotide (nt) strand, called precursor miRNA (pre-miRNA), which retains its hairpin structure. Pre-miRNA is exported, and in the cytoplasm it is recognized and processed by an RNase III enzyme, DICER. This results in two ~22-nt mature miRNAs, which can then be incorporated into the RNA-induced silencing complex (RISC). The pathways forming piRNAs are not yet elucidated. But piRNAs are likely derived from long single-stranded RNAs transcribed in the nucleus. Once in the cytoplasm the precursor piRNAs are then processed into ~30-nt piRNAs. It is known that repeat-associated piRNAs are derived from transcripts of repetitive elements (eg, transposons), and PIWIL2 and PIWIL4 mediate the so-called “ping-pong” amplification loop to generate a large quantity of repeat-associated piRNAs, which can then suppress the expression of those “mother” repetitive elements through methylation of these loci. How non–repeat-associated piRNAs are produced remains unknown.

Figure 6

An overview of posttranscriptional fate control of messenger RNAs (mRNA). Splicing and the addition of the 59 cap and 39 poly(A) tail occur simultaneously and are executed by spliceosomes and heterogeneous nuclear ribonucleoprotein (hnRNP) complexes in the nucleus. Transcripts exported from the nucleus are often associated with hnRNPs. Once in the cytoplasm, the transcripts and their recruited RNA-binding proteins (RBP) are usually first directed to processing or sorting ribonuclear protein particles (RNP; eg, stress granules, p-granules, chromatoid bodies, etc). From there, the transcripts can be either transported and loaded onto polyribosomes for translation, or directed to storage RNPs (RNA operons like Hu antigen R [HUR]/Elav1 granules) for future translation. Transcripts that are no longer needed are directed to the exosomes for degradation.