Light regulates widespread plant alternative polyadenylation through the chloroplast.

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

Iron restriction increases the expression of a cytotoxic cysteine proteinase TvCP2 by a novel mechanism of tvcp2 mRNA alternative polyadenylation in Trichomonas vaginalis.

Trichomonas vaginalis TvCP2 (TVAG_057000) is a cytotoxic cysteine proteinase (CP) expressed under iron-limited conditions. This work aimed to identify one of the mechanisms of tvcp2 gene expression regulation by iron at the posttranscriptional level. We checked tvcp2 mRNA stability under both iron-restricted (IR) and high iron (HI) conditions in the presence of actinomycin D. Greater stability of the tvcp2 mRNA under the IR than in HI conditions was observed, as expected. In silico analysis of the 3' regulatory region showed the presence of two putative polyadenylation signals in the tvcp2 transcript. By 3'-RACE assays, we demonstrated the existence of two isoforms of the tvcp2 mRNA with different 3'-UTR that resulted in more TvCP2 protein under IR than in HI conditions detected by WB assays. Additionally, we searched for homologs of the trichomonad polyadenylation machinery by an in silico analysis in the genome database, TrichDB. 16 genes that encode proteins that could be part of the trichomonad polyadenylation machinery were found. qRT-PCR assays showed that most of these genes were positively regulated by iron. Thus, our results show the presence of alternative polyadenylation as a novel iron posttranscriptional regulatory mechanism in T. vaginalis for the tvcp2 gene expression.

Alternatively polyadenylated calpastatin transcripts in bovine muscles / Transcriptos alternativamente poliadenilados de calpastatina en músculos de bovino

Calpastatin activity has a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditioning. The regulation of calpastatin (CAST) expression is highly complex, the gene has four putative promoters and at least three different polyadenylation sites, and it is also alternatively spliced. We investigated the presence of alternative polyadenylation (APA) isoforms of CAST transcripts in three muscles (infraspinatus, triceps brachii and semitendinosus) of two bovine breeds (Angus and Brahman). The 3´ RACE-PCR was used to specifically amplify the different APA sites. The amplified fragments were cloned and sequenced. Sequencing confirmed the existence of three expected polyadenylation sites corresponding to short, medium and long polyadenylated transcripts. Also, transcripts with a novel APA site were found in the three muscles of both breeds. Because the same APAs isoforms were found between muscles and breeds, we could hypothesize a possible contribution to the relative abundance of different isoforms, probably in coordination with promoter preference and alternative splicing. This knowledge would be useful in the design of future experiments to analyze differential expression of CAST isoforms and their contribution to the definition of beef tenderness.

La actividad de la calpastatina tiene un rol clave en el proceso de tiernización postmortem de la carne durante su almacenamiento refrigerado. La regulación de la expresión de calpastatina (CAST) es altamente compleja; el gen tiene cuatro potenciales promotores, diferentes sitios de poliadenilación de transcriptos y también splicing alternativo. En este trabajo se investiga la presencia de isoformas de transcriptos de CAST alternativamente poliadenilados (APA) en tres músculos (infraspinatus, triceps brachii y semitendinosus) de dos razas bovinas (Angus y Brahman). Se utilizó la técnica de 3´ RACE-PCR para amplificar específicamente los diferentes sitios APA. Los fragmentos amplificados fueron clonados y secuenciados. La secuenciación confirmó la existencia de tres sitios de poliadenilación conocidos. Un nuevo sitio APA fue identificado en transcriptos de los tres músculos y en ambas razas. Dado que cualitativamente no hubo variación en la presencia de isoformas definidas por APA entre músculos y razas de terneza contrastante, podría hipotetizarse una posible contribución a la abundancia relativa de distintas isoformas, probablemente en forma coordinada con la elección de promotores y el splicing alternativo. Este nuevo conocimiento podría ser de utilidad para el diseño de experimentos de análisis de expresión diferencial de isoformas de calpastatina, para ponderar la contribución de las mismas a las variaciones en terneza de la carne.