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.

Molecular targets for Chagas disease: validation, challenges and lead compounds for widely exploited targets.

INTRODUCTION: Chagas disease (CD) imposes social and economic burdens, yet the available treatments have limited efficacy in the disease's chronic phase and cause serious adverse effects. To address this challenge, target-based approaches are a possible strategy to develop new, safe, and active treatments for both phases of the disease. AREAS COVERED: This review delves into target-based approaches applied to CD drug discovery, emphasizing the studies from the last five years. We highlight the proteins cruzain (CZ), trypanothione reductase (TR), sterol 14 α-demethylase (CPY51), iron superoxide dismutase (Fe-SOD), proteasome, cytochrome b (Cytb), and cleavage and polyadenylation specificity factor 3 (CPSF3), chosen based on their biological and chemical validation as drug targets. For each, we discuss its biological relevance and validation as a target, currently related challenges, and the status of the most promising inhibitors. EXPERT OPINION: Target-based approaches toward developing potential CD therapeutics have yielded promising leads in recent years. We expect a significant advance in this field in the next decade, fueled by the new options for Trypanosoma cruzi genetic manipulation that arose in the past decade, combined with recent advances in computational chemistry and chemical biology.

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.

Unraveling the relevance of the polyadenylation factor EhCFIm25 in Entamoeba histolytica through proteomic analysis.

We recently reported that silencing of the polyadenylation factor EhCFIm25 in Entamoeba histolytica, the protozoan which causes human amoebiasis, affects trophozoite proliferation, death, and virulence, suggesting that EhCFIm25 may have potential as a new biochemical target. Here, we performed a shotgun proteomic analysis to identify modulated proteins that could explain this phenotype. Data are available via ProteomeXchange with identifier PXD027784. Our results revealed changes in the abundance of 75 proteins. Interestingly, STRING analysis, functional GO-term annotations, KEGG analyses, and literature review showed that modulated proteins are mainly related to glycolysis and carbon metabolism, cytoskeleton dynamics, and parasite virulence, as well as gene expression and protein modifications. Further studies are needed to confirm the hypotheses emerging from this proteomic analysis, to thereby acquire a comprehensive view of the molecular mechanisms involved.

Advances in the Bioinformatics Knowledge of mRNA Polyadenylation in Baculovirus Genes.

Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3' end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3' ends from 180 baculovirus genomes.

Plant 3' Regulatory Regions From mRNA-Encoding Genes and Their Uses to Modulate Expression.

Molecular biotechnology has made it possible to explore the potential of plants for different purposes. The 3' regulatory regions have a great diversity of cis-regulatory elements directly involved in polyadenylation, stability, transport and mRNA translation, essential to achieve the desired levels of gene expression. A complex interaction between the cleavage and polyadenylation molecular complex and cis-elements determine the polyadenylation site, which may result in the choice of non-canonical sites, resulting in alternative polyadenylation events, involved in the regulation of more than 80% of the genes expressed in plants. In addition, after transcription, a wide array of RNA-binding proteins interacts with cis-acting elements located mainly in the 3' untranslated region, determining the fate of mRNAs in eukaryotic cells. Although a small number of 3' regulatory regions have been identified and validated so far, many studies have shown that plant 3' regulatory regions have a higher potential to regulate gene expression in plants compared to widely used 3' regulatory regions, such as NOS and OCS from Agrobacterium tumefaciens and 35S from cauliflower mosaic virus. In this review, we discuss the role of 3' regulatory regions in gene expression, and the superior potential that plant 3' regulatory regions have compared to NOS, OCS and 35S 3' regulatory regions.

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.

Expression of zebrafish cpsf6 in embryogenesis and role of protein domains on subcellular localization.

CPSF6 is a component of the CFIm complex, involved in mRNA 3'end processing. Despite increasing interest on this protein as a consequence of proposed roles in cancer and HIV infection, several aspects of CPSF6 biological function are poorly understood. In this work we studied the expression of the zebrafish ortholog cpsf6 in early stages of embryo development. Quantitative RT-PCR studies showed that zebrafish cpsf6 mRNA is maternally inherited and that its concentration markedly decreases during early development. We found a generalized distribution of cpsf6 mRNA in early stages through whole mount hybridization experiments. By performing Western blot, we also found a decrease in zebrafish Cpsf6 levels during development. Our analysis of the subcellular localization of this protein using a heterologous system showed a distinct pattern characterized by the presence of nuclear foci. We also studied the relevance of different protein domains on subcellular localization, showing that the C-terminal domain is critical for nuclear localization. Collectively, our results showed that cpsf6 expression changes during early development and that the subcellular localization of the protein is similar to that of the human ortholog.

Posttranscriptional actions of triiodothyronine on Tshb expression in TαT1 cells: New insights into molecular mechanisms of negative feedback.

Rapid actions of triiodothyronine (T3) on thyrotropin (TSH) synthesis and secretion have been described in hypothyroid male rats. However, the molecular mechanisms remain unknown. TαT1 cells, a thyrotroph cell line, was used herein to characterize the possible non-genomic actions of T3 on the expression of alpha (Cga) and Tshb genes, and the posttranscriptional processing and translation of both transcripts. The involvement of αVß3 integrin was also assessed. T3 quickly reduced Tshb mRNA content, poly(A) tail length and its association with ribosomes. The effect of T3 on Tshb gene expression was detected even in the presence of a transcription inhibitor. The decrease in Tshb mRNA content and polyadenylation depend on T3 interaction with αVß3 integrin, while T3 reduced Cga mRNA content by transcriptional action. The translational rate of both transcripts was reduced by a mechanism, which does not depend on T3-αVß3 integrin interaction. Results indicate that, in parallel with the inhibitory transcriptional action in Cga and Tshb gene expression, T3 rapidly triggers additional posttranscriptional mechanisms, reducing the TSH synthesis. These non-genomic actions partially depend on T3-αVß3 integrin interaction at the plasma membrane of thyrotrophs and add new insights to the molecular mechanisms involved in T3 negative feedback loop.

Importance of amino acids Leu135 and Tyr236 for the interaction between EhCFIm25 and RNA: a molecular dynamics simulation study.

The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.

Life and Death of mRNA Molecules in Entamoeba histolytica.

In eukaryotic cells, the life cycle of mRNA molecules is modulated in response to environmental signals and cell-cell communication in order to support cellular homeostasis. Capping, splicing and polyadenylation in the nucleus lead to the formation of transcripts that are suitable for translation in cytoplasm, until mRNA decay occurs in P-bodies. Although pre-mRNA processing and degradation mechanisms have usually been studied separately, they occur simultaneously and in a coordinated manner through protein-protein interactions, maintaining the integrity of gene expression. In the past few years, the availability of the genome sequence of Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, coupled to the development of the so-called "omics" technologies provided new opportunities for the study of mRNA processing and turnover in this pathogen. Here, we review the current knowledge about the molecular basis for splicing, 3' end formation and mRNA degradation in amoeba, which suggest the conservation of events related to mRNA life throughout evolution. We also present the functional characterization of some key proteins and describe some interactions that indicate the relevance of cooperative regulatory events for gene expression in this human parasite.

Silencing the cleavage factor CFIm25 as a new strategy to control Entamoeba histolytica parasite.

The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.

Fibro/adipogenic progenitors safeguard themselves: a novel mechanism to reduce fibrosis is discovered.

PDGFRα regulates several cellular processes, and exacerbated PDGF signaling cause fibrosis in different tissues. Different research groups have shown that fibro/adipogenic progenitors (FAPs) are responsible for pathological fibrosis found in skeletal muscle disorders. Rando's Lab describes that an intronic polyadenylation of Pdgfra regulates FAPs activity, and therefore fibrosis. This discovery opens a new potential target for treating fibrosis.

Amino acid residues Leu135 and Tyr236 are required for RNA binding activity of CFIm25 in Entamoeba histolytica.

Pre-mRNA 3' end processing in the nucleus is essential for mRNA stability, efficient nuclear transport, and translation in eukaryotic cells. In Human, the cleavage/polyadenylation machinery contains the 25 kDa subunit of the Cleavage Factor Im (CFIm25), which specifically recognizes two UGUA elements and regulates the assembly of polyadenylation factors, poly(A) site selection and polyadenylation. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, EhCFIm25 has been reported as a RNA binding protein that interacts with the Poly(A) Polymerase. Here, we follow-up with the study of EhCFIm25 to characterize its interaction with RNA. Using in silico strategy, we identified Leu135 and Tyr236 in EhCFIm25 as conserved amino acids among CFIm25 homologues. We therefore generated mutant EhCFIm25 proteins to investigate the role of these residues for RNA interaction. Results showed that RNA binding activity was totally abrogated when Leu135 and Tyr236 were replaced with Ala residue, and Tyr236 was changed for Phe. In contrast, RNA binding activity was less affected when Leu135 was substituted by Thr. Our data revealed for the first time -until we know-the functional relevance of the conserved Leu135 and Tyr236 in EhCFIm25 for RNA binding activity. They also gave some insights about the possible chemical groups that could be interacting with the RNA molecule.

Controle molecular do acúmulo, tradução e degradação de mRNA em oócitos e embriões / Molecular control of accumulation, translation and degradation of mRNA in oocytes and embryos

A competência oocitária para suportar os posteriores estágios do desenvolvimento depende não somenteda correta segregação cromossômica e de transformações citoesqueléticas, mas, principalmente, da adequadatranscrição e estoque de mRNAs cruciais ao desenvolvimento e à viabilidade celular. Com a retomada dameiose, no entanto, embora o oócito mantenha a capacidade de tradução gênica e de síntese proteica, suaatividade transcricional é interrompida, sendo restabelecida apenas com a ativação do genoma embrionário.Deste modo, todo mRNA materno mobilizado durante maturação, expansão do cumulus, fertilização eembriogênese inicial deve ser sintetizado e estocado, em sua forma traducionalmente inativa, nos oócitosmantidos no estágio diplóteno da prófase I. Complexos mecanismos regulatórios, os quais envolvem apoliadenilação e a desadenilação do mRNA, estão implicados no processo de ativação e silenciamento tantotranscricional quanto traducional. Assim, dada à relevância do tema, esta revisão se propõe a abordar osprincipais eventos moleculares envolvidos no controle da expressão, do estoque, da tradução e da degradação detranscritos maternos imprescindíveis ao desenvolvimento oocitário e embrionário.

The oocyte competence to support the later stages of development depends not only on correctchromosome segregation and cytoskeletal changes, but mainly, the proper transcription and storage of mRNAscritical to the cellular development and viability. However, with the resumption of meiosis, although the oocytekept the ability of gene translation and protein synthesis, its transcriptional activity is interrupted and restoredonly with embryonic genome activation. Thus, all maternal mRNA mobilized during maturation, cumulusexpansion, fertilization and early embryogenesis must be synthesized and stored, in its translationally inactiveform, in oocytes kept in the diplotene stage of prophase I. Complex regulatory mechanisms which involvedeadenylation and polyadenylation of mRNA are involved in this process of activation and silencing astranscriptional as translational. So, due to the importance of the topic, this review proposes to discuss the mainmolecular events involved in the control of expression, storage, translation and degradation of maternaltranscript essential to the oocyte and embryo development.

Controle molecular do acúmulo, tradução e degradação de mRNA em oócitos e embriões / Molecular control of accumulation, translation and degradation of mRNA in oocytes and embryos

A competência oocitária para suportar os posteriores estágios do desenvolvimento depende não somenteda correta segregação cromossômica e de transformações citoesqueléticas, mas, principalmente, da adequadatranscrição e estoque de mRNAs cruciais ao desenvolvimento e à viabilidade celular. Com a retomada dameiose, no entanto, embora o oócito mantenha a capacidade de tradução gênica e de síntese proteica, suaatividade transcricional é interrompida, sendo restabelecida apenas com a ativação do genoma embrionário.Deste modo, todo mRNA materno mobilizado durante maturação, expansão do cumulus, fertilização eembriogênese inicial deve ser sintetizado e estocado, em sua forma traducionalmente inativa, nos oócitosmantidos no estágio diplóteno da prófase I. Complexos mecanismos regulatórios, os quais envolvem apoliadenilação e a desadenilação do mRNA, estão implicados no processo de ativação e silenciamento tantotranscricional quanto traducional. Assim, dada à relevância do tema, esta revisão se propõe a abordar osprincipais eventos moleculares envolvidos no controle da expressão, do estoque, da tradução e da degradação detranscritos maternos imprescindíveis ao desenvolvimento oocitário e embrionário.(AU)

The oocyte competence to support the later stages of development depends not only on correctchromosome segregation and cytoskeletal changes, but mainly, the proper transcription and storage of mRNAscritical to the cellular development and viability. However, with the resumption of meiosis, although the oocytekept the ability of gene translation and protein synthesis, its transcriptional activity is interrupted and restoredonly with embryonic genome activation. Thus, all maternal mRNA mobilized during maturation, cumulusexpansion, fertilization and early embryogenesis must be synthesized and stored, in its translationally inactiveform, in oocytes kept in the diplotene stage of prophase I. Complex regulatory mechanisms which involvedeadenylation and polyadenylation of mRNA are involved in this process of activation and silencing astranscriptional as translational. So, due to the importance of the topic, this review proposes to discuss the mainmolecular events involved in the control of expression, storage, translation and degradation of maternaltranscript essential to the oocyte and embryo development.(AU)