TauCstF-64 Mediates Correct mRNA Polyadenylation and Splicing of Activator and Repressor Isoforms of the Cyclic AMP-Responsive Element Modulator (CREM) in Mouse Testis.

Spermatogenesis is coordinated by the spatial and temporal expression of many transcriptional and posttranscriptional factors. The cyclic AMP-responsive element modulator (CREM) gene encodes both activator and repressor isoforms that act as transcription factors to regulate spermiogenesis. We found that the testis-expressed paralog of CstF-64, tauCstF-64 (gene symbol Cstf2t), is involved in a polyadenylation site choice switch of Crem mRNA and leads to an overall decrease of the Crem mRNAs that are generated from internal promoters in Cstf2t(-/-) mice. More surprisingly, loss of tauCstF-64 also leads to alternative splicing of Crem exon 4, which contains an important activation domain. Thus, testis-specific CREMtau2 isoform protein levels are reduced in Cstf2t(-/-) mice. Consequently, expression of 15 CREM-regulated genes is decreased in testes of Cstf2t(-/-) mice at 25 days postpartum. These effects might further contribute to the infertility phenotype of these animals. This demonstrates that tauCstF-64 is an important stage-specific regulator of Crem mRNA processing that modulates the spatial and temporal expression of downstream stage-specific genes necessary for the proper development of sperm in mice.

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns.

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

ELAV proteins along evolution: back to the nucleus?

The complex interplay of post-transcriptional regulatory mechanisms mediated by RNA-binding proteins (RBP) at different steps of RNA metabolism is pivotal for the development of the nervous system and the maintenance of adult brain activities. In this review, we will focus on the highly conserved ELAV gene family encoding for neuronal-specific RBPs which are necessary for proper neuronal differentiation and important for synaptic plasticity process. In the evolution from Drosophila to man, ELAV proteins seem to have changed their biological functions in relation to their different subcellular localization. While in Drosophila, they are localized in the nuclear compartment of neuronal cells and regulate splicing and polyadenylation, in mammals, the neuronal ELAV proteins are mainly present in the cytoplasm where they participate in regulating mRNA target stability, translation and transport into neurites. However, recent data indicate that the mammalian ELAV RBPs also have nuclear activities, similarly to their fly counterpart, being them able to continuously shuttle between the cytoplasm and the nucleus. Here, we will review and comment on all the biological functions associated with neuronal ELAV proteins along evolution and will show that the post-transcriptional regulatory network mediated by these RBPs in the brain is highly complex and only at an initial stage of being fully understood. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'.

Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64.

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3' UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.

Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV.

The molecular chaperon MRJ (DNAJB6) exhibits two splice isoforms that have different roles in human viral infection, but the regulatory mechanism of MRJ isoform expression is yet unclear. In this study, we show that reduction of the polyadenylation factor CstF64 was correlated with the increase of the MRJ large isoform (MRJ-L) in human macrophages and elucidate the mechanism underlying CstF64-modulated MRJ isoform expression. Moreover, we exploited an antisense strategy targeting MRJ-L for virus replication. A morpholino oligonucleotide complementary to the 5' splice site of MRJ intron 8 downregulated MRJ-L expression and suppressed the replication of not only HIV-1 but also respiratory syncytial virus (RSV). We demonstrated that downregulation of the MRJ-L level reduced HIV-1 replication as well as the subgenomic mRNA and viral production of RSV. The present findings that two human health-threatening viruses take advantage of MRJ-L for infection suggest MRJ-L as a potential target for broad-spectrum antiviral strategy.

Polyadenylation site-specific differences in the activity of the neuronal ßCstF-64 protein in PC-12 cells.

Recent genome-wide analyses have implicated alternative polyadenylation - the process of regulated mRNA 3' end formation - as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, ßCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that ßCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for ßCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that ßCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that ßCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the ß-adducin mRNA. Notably, we demonstrate that the activity of ßCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the ßCstF-64 protein. Our data address the polyadenylation functions of ßCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.