Clinton C. MacDonald

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.

Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

Polyadenylation, the process of eukaryotic mRNA 3′ end formation, is essential for gene expression and cell viability. Polyadenylation of male germ cell mRNAs is unusual, exhibiting increased alternative polyadenylation, decreased AAUAAA polyadenylation signal use, and reduced downstream sequence element dependence. CstF-64, the RNA-binding component of the cleavage stimulation factor (CstF), interacts with pre-mRNAs at sequences downstream of the cleavage site. In mammalian testes, meiotic XY-body formation causes suppression of X-linked CstF-64 expression during pachynema. Consequently, an autosomal paralog, τCstF-64 (gene name Cstf2t), is expressed during meiosis and subsequent haploid differentiation. Here we show that targeted disruption of Cstf2t in mice causes aberrant spermatogenesis, specifically disrupting meiotic and postmeiotic development, resulting in male infertility resembling oligoasthenoteratozoospermia. Furthermore, the Cstf2t mutant phenotype displays variable expressivity such that spermatozoa show a broad range of defects. The overall phenotype is consistent with a requirement for τCstF-64 in spermatogenesis as indicated by the significant changes in expression of thousands of genes in testes of Cstf2t−/− mice as measured by microarray. Our results indicate that, although the infertility in Cstf2t−/− males is due to low sperm count, multiple genes controlling many aspects of germ-cell development depend on τCstF-64 for their normal expression. Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes.

The Gene for a Variant Form of the Polyadenylation Protein CstF-64 Is on Chromosome 19 and Is Expressed in Pachytene Spermatocytes in Mice

Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999)Proc. Natl. Acad Sci. U. S. A. 96, 6763–6768). Previously, we demonstrated the presence of two distinct forms of theM r 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somaticM r 64,000 form and a variantM r 70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form “τ CstF-64,” and we describe here the cloning and characterization of the mouse τCstF-64 cDNA, which maps to chromosome 19. The mouse τCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with τCstF-64 from testis. Features of mτCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro → Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.

Tissue-specific mechanisms of alternative polyadenylation: testis, brain, and beyond.

Changing the position of the poly(A) tail in an mRNA—alternative polyadenylation—is an important mechanism to increase the diversity of gene expression, especially in metazoans. Alternative polyadenylation often occurs in a tissue‐ or developmental stage‐specific manner and can significantly affect gene activity by changing the protein product generated, the stability of the transcript, its localization, or its translatability. Despite the important regulatory effects that alternative polyadenylation have on gene expression, only a sparse few examples have been mechanistically characterized. Here, we review the known mechanisms for the control of alternative polyadenylation, catalog the tissues that demonstrate a propensity for alternative polyadenylation, and focus on the proteins that are known to regulate alternative polyadenylation in specific tissues. We conclude that the field of alternative polyadenylation remains in its infancy, with possibilities for future investigation on the horizon. Given the profound effect alternative polyadenylation can have on gene expression and human health, improved understanding of alternative polyadenylation could lead to numerous advances in control of gene activity. Copyright

Overexpression of the CstF-64 and CPSF-160 Polyadenylation Protein Messenger RNAs in Mouse Male Germ Cells

Abstract Messenger RNAs for several components of the transcriptional apparatus are greatly overexpressed in postmeiotic male germ cells in rodents (Schmidt and Schibler, Development 1995; 121:2373–2383). Because of the tight coupling of polyadenylation and transcription, we examined expression in germ cells of mRNAs for key polyadenylation factors. The mRNA for the 64 000 Mr subunit of the cleavage stimulation factor (CstF-64) was expressed at least 250-fold greater in mouse testicular RNA than in liver RNA. RNA blot analysis showed that the mRNA for the 160 000 Mr subunit of the cleavage and polyadenylation specificity factor was similarly overexpressed, as was the mRNA for the large subunit of RNA polymerase II. General transcription factors, such as the TATA-binding protein and transcription factor IIH, and splicing factors, such as components of the small nuclear ribonucleoproteins, were also expressed in meiotic and postmeiotic germ cells. The X-linked CstF-64 protein is expressed before and after but not during meiosis in the mouse (Wallace et al., Proc Natl Acad Sci U S A 1999; 96:6763–6768), which suggests that overexpression of mRNA transcription and processing factors plays an essential role in postmeiotic germ cell mRNA metabolism.

Developmental Distribution of the Polyadenylation Protein CstF-64 and the Variant τCstF-64 in Mouse and Rat Testis

Abstract Messenger RNA polyadenylation is one of the processes that control gene expression in all eukaryotic cells and tissues. In mice, two forms of the regulatory polyadenylation protein CstF-64 are found. The gene Cstf2 on the X chromosome encodes this form, and it is expressed in all somatic tissues. The second form, τCstF-64 (encoded by the autosomal gene Cstf2t), is expressed in a more limited set of tissues and cell types, largely in meiotic and postmeiotic male germ cells and, to a smaller extent, in brain. We report here that whereas CstF-64 and τCstF-64 expression in rat tissues resembles their expression in mouse tissues, significant differences also are found. First, unlike in mice, in which CstF-64 was expressed in postmeiotic round and elongating spermatids, rat CstF-64 was absent in those cell types. Second, unlike in mice, τCstF-64 was expressed at significant levels in rat liver. These differences in expression suggest interesting differences in X-chromosomal gene expression between these two rodent species.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems

BackgroundAlternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs.ResultsWe discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that βCstF-64 is expressed in neurons but not glia.ConclusionThis is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that βCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs.

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, tauCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between tauCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of tauCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and tauCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than tauCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal alpha-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is mportant in RNA sequence recognition. This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.

The Hinge Domain of the Cleavage Stimulation Factor Protein CstF-64 Is Essential for CstF-77 Interaction, Nuclear Localization, and Polyadenylation

Because polyadenylation is essential for cell growth, in vivo examination of polyadenylation protein function has been difficult. Here we describe a new in vivo assay that allows structure-function assays on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. In this assay (the stem-loop luciferase assay for polyadenylation, SLAP), expression of a luciferase pre-mRNA with a modified downstream sequence element was made dependent upon co-expression of an MS2-CstF-64 fusion protein. We show here that SLAP accurately reflects CstF-64-dependent polyadenylation, confirming the validity of this assay. Using SLAP, we determined that CstF-64 domains involved in RNA binding, interaction with CstF-77 (the “Hinge” domain), and coupling to transcription are critical for polyadenylation. Further, we showed that the Hinge domain is necessary for CstF-64 interaction with CstF-77 and consequent nuclear localization, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.

The τCstF-64 Polyadenylation Protein Controls Genome Expression in Testis

The τCstF-64 polyadenylation protein (gene symbol Cstf2t) is a testis-expressed orthologue of CstF-64. Mice in which Cstf2t was knocked out had a phenotype that was only detected in meiotic and postmeiotic male germ cells, giving us the opportunity to examine CstF-64 function in an isolated developmental system. We performed massively parallel clonally amplified sequencing of cDNAs from testes of wild type and Cstf2t−/− mice. These results revealed that loss of τCstF-64 resulted in large-scale changes in patterns of genome expression. We determined that there was a significant overrepresentation of RNAs from introns and intergenic regions in testes of Cstf2t−/− mice, and a concomitant use of more distal polyadenylation sites. We observed this effect particularly in intronless small genes, many of which are expressed retroposons that likely co-evolved with τCstF-64. Finally, we observed overexpression of long interspersed nuclear element (LINE) sequences in Cstf2t−/− testes. These results suggest that τCstF-64 plays a role in 3′ end determination and transcription termination for a large range of germ cell-expressed genes.