Alternative splicing is a regulatory mechanism that allows amplification of the complexity and plasticity of the genome through the generation of different isoforms from a single gene. Although it occurs in all tissues, brain and testis display the highest levels of alternative splicing events, which participate to the differentiation of highly specialized cells like neurons and gametes. In a pre-mRNA, the choice of the exons to be spliced is governed by a delicate balance between two classes of splicing factors that act antagonistically: hnRNP proteins (splicing inhibitors) and SR proteins (splicing activators). Since many changes in alternative splicing events occur during germ cell differentiation, we tested whether changes in the expression of specific hnRNP and SR proteins trigger these events. Western blot analysis showed that many hnRNPs and SR proteins are highly modulated during testis development. For example, there is a switch between hnRNP I isoforms, with the somatic protein (PTB) decreasing after 20 dpp and the concomitant increase in the neuron-specific nPTB isoform. In addition, we observed a general decrease in SR proteins and the maintenance of the cellular levels of their antagonist hnRNPs. These results were confirmed by isolating germ cells at different stages of development: mitotic spermatogonia, meiotic spermatocytes and haploid spermatids. Since hnRNPs normally inhibit alternative splicing, these results could explain why in the testis many events of exon skipping occur during germ cell differentiation. We are currently testing this hypothesis by using minigenes that recapitulate germ cell-specific alternative splicing events. Since several splicing factors participate also to cytoplasmic regulation of mRNAs, we performed immunofluorence analyses to determine their subcellular localization in differentiating germ cells. We found that ASF/SF2 and hnRNP F/H localize in the cytoplasm in meiotic spermatocytes. Moreover, sucrose fractionation experiments indicated that these splicing factors associate with polyribosomes engaged in translation in meiotic cells. We are currently performing functional analysis to determine if these splicing factors regulate translation of specific mRNAs during germ cell differentiation.
Regulation of gene expression by alternative spicing during germ cell differentiation
Paronetto MP;
2008-01-01
Abstract
Alternative splicing is a regulatory mechanism that allows amplification of the complexity and plasticity of the genome through the generation of different isoforms from a single gene. Although it occurs in all tissues, brain and testis display the highest levels of alternative splicing events, which participate to the differentiation of highly specialized cells like neurons and gametes. In a pre-mRNA, the choice of the exons to be spliced is governed by a delicate balance between two classes of splicing factors that act antagonistically: hnRNP proteins (splicing inhibitors) and SR proteins (splicing activators). Since many changes in alternative splicing events occur during germ cell differentiation, we tested whether changes in the expression of specific hnRNP and SR proteins trigger these events. Western blot analysis showed that many hnRNPs and SR proteins are highly modulated during testis development. For example, there is a switch between hnRNP I isoforms, with the somatic protein (PTB) decreasing after 20 dpp and the concomitant increase in the neuron-specific nPTB isoform. In addition, we observed a general decrease in SR proteins and the maintenance of the cellular levels of their antagonist hnRNPs. These results were confirmed by isolating germ cells at different stages of development: mitotic spermatogonia, meiotic spermatocytes and haploid spermatids. Since hnRNPs normally inhibit alternative splicing, these results could explain why in the testis many events of exon skipping occur during germ cell differentiation. We are currently testing this hypothesis by using minigenes that recapitulate germ cell-specific alternative splicing events. Since several splicing factors participate also to cytoplasmic regulation of mRNAs, we performed immunofluorence analyses to determine their subcellular localization in differentiating germ cells. We found that ASF/SF2 and hnRNP F/H localize in the cytoplasm in meiotic spermatocytes. Moreover, sucrose fractionation experiments indicated that these splicing factors associate with polyribosomes engaged in translation in meiotic cells. We are currently performing functional analysis to determine if these splicing factors regulate translation of specific mRNAs during germ cell differentiation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
