Androgen-responsive microRNAs in mouse Sertoli cells.

Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs--small, non-coding RNAs--are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.

Interaction between microRNAs and actin-associated protein Arpc5 regulates translational suppression during male germ cell differentiation.

Decoupling of transcription and translation during postmeiotic germ cell differentiation is critical for successful spermatogenesis. Here we establish that the interaction between microRNAs and actin-associated protein Arpc5 sets the stage for an elaborate translational control mechanism by facilitating the sequestration of germ cell mRNAs into translationally inert ribonucleoprotein particles until they are later translated. Our studies reveal that loss of microRNA-dependent regulation of Arpc5, which controls the distribution of germ cell mRNAs between translationally active and inactive pools, results in abnormal round spermatid differentiation and impaired fertility. Interestingly, Arpc5 functions as a broadly acting translational suppressor, as it inhibits translation initiation by blocking 80S formation and facilitates the transport of mRNAs to chromatoid/P bodies. These findings identify a unique role for actin-associated proteins in translational regulation, and suggest that mRNA-specific and general translational control mechanisms work in tandem to regulate critical germ cell differentiation events and diverse somatic cell functions.

Genomic loss of tumor suppressor miRNA-204 promotes cancer cell migration and invasion by activating AKT/mTOR/Rac1 signaling and actin reorganization.

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis.

A thorough understanding of the events during mammalian spermatogenesis requires studying specific molecular signatures of individual testicular cell populations as well as their interaction in co-cultures. However, most purification techniques to isolate specific testicular cell populations are time-consuming, require large numbers of animals, and/or are only able to isolate a few cell types. Here we describe a cost-effective and timesaving approach that uses a single protocol to enrich multiple testicular cell populations (Sertoli, Leydig, and several spermatogenic cell populations) from as few as one mouse. Our protocol combines rigorous enzymatic digestion of seminiferous tubules with counter-current centrifugal elutriation, yielding specific testicular cell populations with >80%-95% purity.

Role of ß-catenin in post-meiotic male germ cell differentiation.

Though roles of ß-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that ß-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of ß-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of ß-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that ß-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since ß-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell ß-catenin complex to ß-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.

Nonsense codons trigger an RNA partitioning shift.

T-cell receptor-beta (TCRbeta) genes naturally acquire premature termination codons (PTCs) as a result of programmed gene rearrangements. PTC-bearing TCRbeta transcripts are dramatically down-regulated to protect T-cells from the deleterious effects of the truncated proteins that would otherwise be produced. Here we provide evidence that two responses collaborate to elicit this dramatic down-regulation. One is rapid mRNA decay triggered by the nonsense-mediated decay (NMD) RNA surveillance pathway. We demonstrate that this occurs in highly purified nuclei lacking detectable levels of three different cytoplasmic markers, but containing an outer nuclear membrane marker, suggesting that decay occurs either in the nucleoplasm or at the outer nuclear membrane. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCRbeta transcripts escaping to the cytoplasmic fraction of cells. Analysis of TCRbeta mRNA kinetics after either transcriptional repression or induction suggested that this nonsense codon-induced partitioning shift (NIPS) response is not the result of cytoplasmic NMD but instead reflects retention of PTC(+) TCRbeta mRNA in the nuclear fraction of cells. We identified TCRbeta sequences crucial for NIPS but found that NIPS is not exclusively a property of TCRbeta transcripts, and we identified non-TCRbeta sequences that elicit NIPS. RNA interference experiments indicated that NIPS depends on the NMD factors UPF1 and eIF4AIII but not the NMD factor UPF3B. We propose that NIPS collaborates with NMD to retain and degrade a subset of PTC(+) transcripts at the outer nuclear membrane and/or within the nucleoplasm.

Alternatively spliced T-cell receptor transcripts are up-regulated in response to disruption of either splicing elements or reading frame.

Nonsense mutations create premature termination codons (PTCs), leading to the generation of truncated proteins, some of which have deleterious gain-of-function or dominant-negative activity. Protecting cells from such aberrant proteins is non-sense-mediated decay (NMD), an RNA surveillance pathway that degrades transcripts harboring PTCs. A second response to nonsense mutations is the up-regulation of alternatively spliced transcripts that skip the PTC. This nonsense-associated altered splicing (NAS) response has the potential to rescue protein function, but the mechanism by which it is triggered has been controversial. Some studies suggest that, like NMD, NAS is triggered as a result of nonsense mutations disrupting reading frame, whereas other studies suggest that NAS is triggered when nonsense mutations disrupt exonic splicing enhancers (ESEs). Using T-cell receptor-beta (TCRbeta), which naturally acquires PTCs at high frequency, we provide evidence that both mechanisms act on a single type of mRNA. Mutations that disrupt consensus ESE sites up-regulated an alternatively spliced TCRbeta transcript that skipped the mutations independently of reading frame disruption and the NMD factor UPF1. In contrast, reading frame-disrupting mutations that did not disrupt consensus ESE sites elicited UPF1-dependent up-regulation of the alternatively spliced TCRbeta transcript. Restoration of reading frame prevented this up-regulation. Our results suggest that the response of an mRNA to a nonsense mutation depends on its context.

The nonsense-mediated decay RNA surveillance pathway.

Nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that selectively degrades mRNAs harboring premature termination (nonsense) codons. If translated, these mRNAs can produce truncated proteins with dominant-negative or deleterious gain-of-function activities. In this review, we describe the molecular mechanism of NMD. We first cover conserved factors known to be involved in NMD in all eukaryotes. We then describe a unique protein complex that is deposited on mammalian mRNAs during splicing, which defines a stop codon as premature. Interaction between this exon-junction complex (EJC) and NMD factors assembled at the upstream stop codon triggers a series of steps that ultimately lead to mRNA decay. We discuss whether these proofreading events preferentially occur during a "pioneer" round of translation in higher and lower eukaryotes, their cellular location, and whether they can use alternative EJC factors or act independent of the EJC.

An alternative branch of the nonsense-mediated decay pathway.

The T-cell receptor (TCR) locus undergoes programmed rearrangements that frequently generate premature termination codons (PTCs). The PTC-bearing transcripts derived from such nonproductively rearranged genes are dramatically downregulated by the nonsense-mediated decay (NMD) pathway. Here, we show that depletion of the NMD factor UPF3b does not impair TCRbeta NMD, thereby distinguishing it from classical NMD. Depletion of the related factor UPF3a, by itself or in combination with UPF3b, also has no effect on TCRbeta NMD. Mapping experiments revealed the identity of TCRbeta sequences that elicit a switch to UPF3b dependence. This regulation is not a peculiarity of TCRbeta, as we identified many wild-type genes, including one essential for NMD, that transcribe NMD-targeted mRNAs whose downregulation is little or not affected by UPF3a and UPF3b depletion. We propose that we have uncovered an alternative branch of the NMD pathway that not only degrades aberrant mRNAs but also regulates normal mRNAs, including one that participates in a negative feedback loop controlling the magnitude of NMD.

Nonsense-associated altered splicing: a frame-dependent response distinct from nonsense-mediated decay.

Nonsense-associated altered splicing (NAS) is a putative correction response that upregulates alternatively spliced transcripts that have skipped offending premature termination codons (PTCs). Here, we examined whether NAS has characteristics in common with nonsense-mediated decay (NMD), a surveillance mechanism that degrades PTC-bearing mRNAs. We discovered that although NAS shared the need for a Kozak AUG to define frame, it differed from NMD. NAS was not affected by depletion of the NMD protein hUPF2, and it functioned independently of RNA stabilization. We identified an alternatively spliced transcript acted upon by both NAS and NMD, indicating that these two mechanisms are not mutually exclusive. Our results suggest that NAS and NMD are distinct mechanisms despite being triggered by the same signal.