<p>This review article presents the splicing process during messenger RNA maturation and how it is regulated by different <em>Cis</em>-regulatory RNA-sequence elements and splicing factors. A more detailed description of the process alternative splicing and its importance to the function of genes from the model organism <em>Arabidopsis thaliana</em> is also given. A single eukaryotic gene can by the process alternative splicing (AS) give rise to a number of functionally mature mRNA-molecules, which in turn encodes for structurally and/or functionally different proteins. During the course of evolution, the process alternative splicing has thus shown to be effective in increasing transcriptome and proteome diversity of most eukaryotic organisms. This suggests therefore that the dominant theory in molecular biology, a gene encodes for a protein, needs to be corrected. A future challenge is to determine the function of the proteins obtained from a given gene by alternative splicing.</p>

xiv, 59 p. : ill. (some col.) / Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy (DM). In DM, the lack of properly localized MBNL1 leads to mis-splicing of many pre-mRNAs. The mechanism by which MBNL1 regulates it pre-mRNA targets is not well understood. In order to determine the mechanism by which MBNL1 regulates alternative splicing, a consensus RNA binding motif for Mbl (the <italic>Drosophila</italic> ortholog of MBNL1) and MBNL1 were determined using SELEX (Systematic Evolution of Ligands by Exponential Enrichment). These consensus motifs allowed for the identification of high affinity endogenous sites within pre-mRNAs that are regulated by MBNL1. <italic>In vitro</italic> binding studies showed that MBNL1 bound to RNAs that contained the consensus motif surrounded by pyrimidines. Some of these sites were identified upstream of exon 5 within the <italic>MBNL1</italic> pre-mRNA, and we have shown that MBNL1 auto-regulates the exclusion of exon 5 in HeLa cells. The region of the <italic>MBNL1</italic> gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3' end of intron 4 is non-canonical in that it contains an AAG 3' splice site and a predicted branchpoint that is 141 nucleotides from the 3' splice site. Using a mini-gene that includes exon 4, intron 4, exon 5, intron 5 and exon 6 of <italic>MBNL1</italic>, we show that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branchpoint confirms that branching occurs primarily at the predicted distant branchpoint. Structure probing and footprinting reveal that the highly conserved region between the branchpoint and the 3' splice site is primarily unstructured, and MBNL1 binds within this region of the pre-mRNA, which we have termed the MBNL1 response element. Deletion of the response element eliminates MBNL1 splicing regulation and leads to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing. This dissertation includes previously published co-authored material as well as my recent co-authored material that has been submitted for publication. / Committee in charge: Kenneth Prehoda, Chair; J. Andrew Berglund, Advisor; Victoria J. de Rose, Member; Alice Barkan, Member; Karen Guillemin, Outside Member

Protozoários do gênero Trypanosoma infectam milhões de pessoas todo ano e coletivamente contribuem muito para as misérias humanas, pois são causa de muitas das doenças negligenciadas tropicais. Várias vias metabólicas essenciais são encontradas nesses parasitas tornando-os particularmente atrativos para investigações moleculares. Mecanismos de controle pós-transcricional tem sido alvo de estudo por sua peculiaridade nesses organismos. Nesse cenário, proteínas da classe das poli-A-binding proteins (PABP) possuem função no início da tradução, turnover do mRNA e interação com o 5´-CAP. Nesse trabalho foi identificada a homóloga poli-A binding protein 1 (PABP1) de Trypanosoma brucei. O silenciamento do gene pabp1 revelou que a ausência da proteína é letal ao parasita, comprovando sua essencialidade nesse organismo. Da mesma maneira, na ausência da proteína observou-se erro no processamento do mRNA sugerindo possível função nos eventos de cis e trans splicing. Sua localização subcelular foi avaliada indicando localização citoplasmática, bem como o são suas homólogas. No citoplasma, a proteína apresenta-se em estrutura reticulada, co-localizada com proteínas de retículo endoplasmático. Porém, sob estresse induzido a proteína relocaliza para o compartimento nuclear, indicando ser uma proteína com trânsito núcleo-citoplasma ainda não demonstrada na literatura. As funções identificadas sugerem a existência de um sub-complexo a 3´ do mRNA que acopla poliadenilação e splicing. Além disso, a relocalização nuclear parece ocorrer em resposta a estímulo externo, sugerindo que a relocalização do mRNA para o núcleo pode ser uma estratégia da célula para modular sua resposta gênica frente a variações do ambiente. / Protozoa of the genus Trypanosoma infect millions of people every year and collectively contribute to the human misery by causing several neglected tropical diseases. Several intriguing molecular pathways are found in these parasites also, rendering them particularly attractive for biochemical investigation. This unique eukaryotic cells lack mechanisms to control gene expression at the transcriptional level, they mostly control protein synthesis by posttranscriptional regulation process. Several RNAs and proteins are involved in this process, including poly(A) binding proteins. The poly(A)- binding protein of eukaryotes plays a role in polyadenylation, translation initiation and metabolism of mRNA. In this work the poly(A) binding protein 1 (PABP1) was identified in Trypanosoma brucei. Depletion of TbPABP1 showed its essential role in the procyclic form of the parasite. Immunofluorescence assays showed localization in the cytosolic compartment despite of its functions in cis and trans splicing as shown by RNA analysis of cells free from PABP1. As it was shown in the homologs, PABP1 it´s not only a cytosolic protein but it shuttles between the nucleus and the cytoplasm. Together with the literature, these results suggest an active complex in the 3´ end of the mRNA which works in synchrony with the splicing and capping machinery implying PABP1 as possible link between these processes.

A excisão de sequencias intrônicas dos precursores de mRNAs é um passo crítico durante a expressão gênica eucariótica. Essa reação é catalisada pelo complexo macromolecular denominado spliceossomo, composto por partículas ribonucleoproteicas nucleares (U1, U2, U4/U6, U5 snRNPs), além de inúmeros fatores associados. Em tripanossomatídeos, os genes são transcritos em longas unidades policistrônicas e a reação de SL trans-splicing é requerida para geração de transcritos monocistrônicos maduros. O spliceossomo é uma maquinaria altamente dinâmica e parte das mudanças conformacionais ocorridas neste complexo é mediada por RNA helicases. A RNA helicase/ATPásica Sub2 (em mamífero, UAP56), cujas homólogas foram descritas como membros do complexo TREX (transcrição/exportação do RNA), é essencial na montagem do pré-spliceossomo, além de estudos sugerirem sua participação em outras etapas de montagem desse complexo. Em tripanossomatídeos, a função desta proteína no transporte de mRNA entre núcleo e citoplasma já foi descrita, entretanto, seu envolvimento na reação de splicing permanece indefinido. Neste trabalho, buscou-se estudar esse envolvimento através de técnicas de purificação em tandem e RNA de interferência. A purificação dos parceiros de interação da proteína somada à identificação por espectrometria de massas mostrou que TbSub2 co-purifica com proteínas envolvidas em múltiplas vias do metabolismo do parasito, incluindo proteínas e fatores relacionados ao processamento de mRNA. Além disso, essa proteína é essencial para os parasitos na forma procíclica e sanguínea e apresenta localização nuclear em ambas as linhagens. Análises por qPCR em tempo real e RT-PCR mostraram que o silenciamento de TbSub2 causa defeito na maquinaria de SL trans-splicing mas seu efeito no cis-splicing não é claro. Por fim, foi possível realizar a expressão heteróloga e purificação da proteína TbSub2 recombinante, bem como estudos para avaliar seu estado oligomérico e estabilidade. Dessa forma, foi constatado que essa proteína é estável em diferentes tampões e apresenta estados oligoméricos distintos nas técnicas empregadas nesse trabalho. O estudo aqui apresentado trouxe evidências da participação da proteína TbSub2 na reação de SL trans-splicing em ambas as linhagens, bem como em múltiplas vias do metabolismo do parasito na forma procíclica, contribuindo para uma elucidação das funções dessa proteína em tripanossomatídeos. / The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the macromolecular complex called spliceosome, composed of nuclear ribonucleoprotein particles (U1, U2, U4 / U6, U5 snRNPs), besides numerous associated factors. In trypanosomatids, genes are transcribed in long polycistronic units and the SL trans-splicing reaction is required for the generation of mature monocistronic transcripts. The spliceosome is a highly dynamic machinery and part of this complex\'s conformational changes is regulated by RNA helicases. The RNA helicase / ATPase Sub2 UAP56, in mammalian), whose homologous proteins have been described as members of the TREX complex (transcription / RNA export), is essential in the pre-spliceosome assembly, besides its participation in other assembly steps of this complex, as suggested by other studies. In trypanosomatids, the role of this protein in the nucleus/cytoplasm mRNA transport has already been described, however its involvement in the splicing reaction remains unknown. In this work, we studied this involvement through tandem affinity purification and RNA interference. The purification of this proteins binding partners added to the identification by mass spectrometry showed that TbSub2 co-purifies with proteins involved in multiple metabolism pathways of the parasite, including proteins and factors related to mRNA processing. In addition, this protein is essencial for the parasites in procyclic and bloodstream forms and localizes in the nucleus in both strains. qPCR and RT-PCR analysis showed that the silencing of TbSub2 causes defect in the SL trans-splicing machinery but its effect on cis-splicing is unclear. Finally, it was possible to perform the heterologous expression and purification of recombinant TbSub2 protein, as well as studies to evaluate its oligomeric state and stability. Thus, it was found that this protein is stable in different buffers and presents different oligomeric states in the techniques employed in this work. This study has provided evidence of this helicases participation in the SL trans-splicing reaction in booth strains, as well as in several metabolism pathways of the parasite in prociclic form, contributing to the elucidation of the protein functions in trypanosomatids.

A doença do sono é um dos maiores obstáculos para o desenvolvimento das áreas rurais da África Subsaariana. O diagnóstico positivo da doença do sono, bem como o estágio em que ela se encontra, é essencial perante a severidade da doença e toxicidade dos medicamentos disponíveis para o tratamento. A eficiência do tratamento e diagnóstico depende do conhecimento do ciclo de vida, biologia do parasito e seu metabolismo. Os tripanossomatídeos possuem mecanismos conservados entre si como a expressão gênica, neste contexto o Trypanosoma brucei pode ser considerado um organismo modelo e o estudo do processamento de RNA mensageiro por splicing neste parasito pode ser extrapolado para outros tripanosomatídeos. O processo de excisão dos íntrons e junção dos éxons é chamado splicing, e tanto o cis quanto o trans-splicing são reações de transesterificação realizados pelo spliceosomo, que consiste de 5 partículas nucleares, as snRNPs (small nuclear ribonucleoprotein) U1, U2, U4, U5 e U6 bem como proteínas não específicas de snRNPs. As snRNPs são complexos que consistem de pequenos RNAs ricos em uridina (U snRNAs) unidos fortemente a proteínas. São conhecidas oito proteínas específicas de U5 snRNP em humanos 220K, 200K, 116K, 102K, 100K, 52K, 40K e 15K. Foi mostrado que a U5-15K humana é essencial em diversos pontos da formação do spliceosomo. O objetivo desse trabalho foi a caracterização estrutural e da atividade de autoclivagem da proteína U5-15K de T. brucei. Essa proteína pertencente à família Dim1e é formada por 155 resíduos de aminoácido e massa molecular de 17,7 kDa. A proteína recombinante clonada no vetor de expressão pET SUMO (Invitrogen) foi expressa em E. coli por indução com IPTG e purificada por cromatografia de afinidade por íons metálico em resina de Cobalto. O produto foi usado para testes da atividade de auto-clivagem, contendo ou não inibidores de protease. A proteína pura também foi usada em estudos de suas propriedades em solução por experimentos de DLS, que mostrou uma maior homogeneidade da proteína na presença de MgCl2, DSF, que mostrou a estabilidade da U5-15K em solução com relação ao pH. As mudanças de conformação da estrutura secundária da U5-15K e U5-15K clivada foi estudada por experimentos de CD, que mostraram uma redução na porcentagem de folhas-&beta; na estrutura terciária da U5-15K clivada, e foi construído um modelo tridimensional através da modelagem por homologia, que foi comparado com os resultados obtidos por experimentos de SAXS. Foram realizados ensaios de cristalização em diversas condições provenientes de kits comerciais com a proteína nas formas clivada e não clivada e em diferentes concentrações. Como perspectivas ficam a definição exata do ponto de clivagem por espectrometria de massas, proteólise da alça flexível para novos ensaios de cristalização e estudo das mutações nos possíveis sítios ativos e sítio de clivagem. / Sleep sickness is one the most important public health problem in the Africa and it causative agent, Trypanosma brucei, is an organism model for the study of different conserved process among the trypanosomatids. In trypanosomes, mRNAs are processed by trans-splicing, in which a common spliced leader sequence (SL) is acquired at the 5\' end of the mRNA to yield a mature transcript. RNA splicing is carried out by the spliceosome, which consists of the U1, U2, U4, U5 and U6 U snRNPs particles and non-snRNP proteins. The ribonucleoproteins are complexes that consist of small uridine-rich RNAs (U snRNAs) and interact with common Sm proteins and proteins that are specific for each snRNP. Seven U5 snRNP specific proteins are known in trypanosomes, 220K, 200K, 116K, 102K, Cwc21, 40K e 15K. The spliceosomal protein U5-15K is essential for the parasite viability and various evidences suggest participation of the member in spliceosome assembly. U5-15K presents a conserved domain dim1, its molecular weight is estimated of 17,7 kDa and 155 amino acids residues. In this work, U5-15K was cloned into pET SUMO (Invitrogen), transformed in BL21(DE3)pLysS and recombinant protein was purified by immobilized ion affinity chromatography. It was possible observe that U5-15K undergo self-cleavage, process inhibited by serine and cysteine protease inhibitors. Dynamic Light Scattering (DLS) experiments showed higher protein homogeneity in the presence of MgCl2 and Differential Scanning Fluorimetry (DSF) data demonstrated higher stability at neutral pH. Circular dichroism (CD) spectra obtained using U5-15K native and cleaved suggest a reduction in percentage of &beta;-sheets at cleaved U5-15K secondary structure. Native and cleaved proteins at different concentrations were used in crystallization trials, however, no suitable protein crystals were observed. A tridimensional homology model for U5-15K from Trypanosoma brucei, using as template the human homologue, present a thioredoxin folding, although it has observed a possible central loop not present in the template. We intent to determine the exact cleavage point using mass spectrometry and new crystallization trials will be performed after removal of probable loops by limited proteolysis.

Entre as modificações sofridas pelo transcrito primário de RNA de eucariontes, o splicing é responsável pela colocação lado a lado das sequências expressas alinhando a região codificadora no RNAm. Este mecanismo, descrito na década de 1970, como o responsável pela remoção dos íntrons e junção dos éxons consecutivos, é efetuado por um complexo ribonucleoprotéico conhecido como spliceossomo. O reconhecimento por este complexo dos segmentos definidos como éxons e íntrons depende de diversas sequências presentes no RNA e reconhecidas por ligantes protéicos. A modulação desta interação resulta na geração de diferentes transcritos maduros a partir de um mesmo gene, evento conhecido como splicing alternativo, comum a maioria dos genes humanos e um dos grandes responsáveis pela geração de variabilidade proteômica dos eucariotos e sua complexidade morfo-fisiológica. O splicing alternativo é um importante gerador de diversidade funcional no sistema nervoso central, onde participa da geração de variantes para mais de 80% dos genes. Entre estes está o gene do Retardo Mental do X Frágil (FMR1), cujo transcrito primário pode sofrer splicing alternativo de quatro éxons, produzindo até vinte isoformas diferentes da FMRP. Os objetivos gerais deste projeto foram (i) a análise da expressão do éxon 12 do Fmr1 em córtex cerebral frontal, hipocampo e cerebelo de ratos em E19 e P2; e (ii) a busca por elementos em cis reguladores do splicing do éxon 12 do Fmr1 de rato. Para averiguar os níveis da expressão do éxon 12 do Fmr1, no final do período embrionário e início do pós-natal de rato, foi realizada RT-PCR em tempo real com os tecidos citados acima, em E19 e P2. Observamos significativa inclusão do éxon 12 nos transcritos do Fmr1 no córtex frontal em P2 quando comparado a E19, o que não se relacionou ao aumento geral da expressão do Fmr1. No hipocampo, houve aumento da expressão do conjunto de mensagens do Fmr1 e tendência à exclusão do éxon 12 em P2, quando comparado a E19. Estes dados revelam o córtex cerebral como fonte de proteínas ativadoras do splicing do éxon 12 do Fmr1 e onde se deve buscar pela relevância funcional das isoformas da FMRP expressando este éxon. A busca por elementos reguladores do splicing do éxon 12 se baseiou na avaliação da expressão por RT-PCR de mini-gene de segmento genômico do gene Fmr1 usado para transfectar células C6 (glioma de rato). Estas células demonstraram inclusão preferencial do éxon 12 em seus transcritos superexpressos. Um segundo clone foi gerado com uma deleção a partir do clone original, na região 5 do íntron 12, na qual observamos in silico, elementos ricos em U e C, candidatos a acentuadores da inclusão do éxon 12. A superexpressão deste clone em C6 revelou exclusão preferencial do éxon 12, um padrão invertido em relação ao anteriormente observado. Estes dados indicam o elemento rico em U e C como um forte candidato a acentuar a inclusão do éxon 12 no RNAm do Fmr1. / Splicing is an important hnRNA processing mechanism in eukaryotes, aligning exons in the mRNA. First described in the 1970s, it is performed by a molecular complex named spliceosome, which recognizes RNA sequences in the boundaries between exons and introns. Interaction modulation in the spliceosome results in mature transcripts with varying sizes, a process known as alternative splicing, common to most human genes and the major mechanism leading to proteomic diversity and morphological and functional complexity in eukaryotes. Alternative splicing is very important in generating functional diversity in the central nervous system (CNS), where it takes part in more than 80% of primary transcript processing. Among these is the Fragile Mental Retardation 1 gene (FMR1), which undergoes alternative splicing of four exons creating the possibility of 20 non-redundant FMRP isoforms. The aims of this project were (i) to analyze the expression of rat Fmr1 exon 12 in frontal cerebral cortex, hippocampus, and cerebellum at E19 and P2 days; and (ii) to search for cis-acting elements regulating exon 12 splicing. We performed real-time RT-PCR to examine Fmr1 exon 12 expression, in the above-mentioned CNS structures, between the end of embryonic period and the second postnatal day. We observed significant inclusion of exon 12 in Fmr1 mRNA in frontal cortex at P2 as compared to E19, which was unrelated to general Fmr1 expression increase. At P2 hippocampus there was a significant increase at the expression levels of Fmr1, and a trend to exclude exon 12 from the primary transcript. This data indicates that cerebral cortex is an important source of proteins activating exon 12 splicing, and also a tissue where the functional relevance of FMRP isoforms expressing exon 12 should be regarded. We adopted the mini-gene approach to search for cis elements regulating Fmr1 exon 12 splicing. RTPCR was performed to evaluate C6 (rat glioma) cells overexpressing a clone containing a genomic Fmr1 segment. Transfected cells revealed preferential inclusion of Fmr1 exon 12. A deletion construct lacking the initial bases of intron 12 was generated. The deleted segment harbor U- and C-rich sequences that had been identified in silico in a search for intronic splicing enhancers. Overexpression of the deletion construct in C6 yielded to preferential exclusion of exon 12, as opposed to the expression pattern previously observed in the original clone. Therefore, the U- and C-rich elements at Fmr1 intron 12 are strong candidates to enhance Fmr1 exon 12 splicing.

O splicing é o processo que resulta na remoção dos íntrons e união dos éxons nos genes eucarióticos. Nesse processo diversos elementos em cis e trans estão envolvidos. Além das sequências em cis canônicas (sítios de splicing, sítio de ramificação e trato de polipirimidina), os reguladores de splicing -- sequências curtas localizadas em éxons e íntrons - são considerados de grande importância, pois auxiliam na correta determinação das fronteiras éxons/introns. Através do splicing alternativo pode-se gerar grande diversidade de transcritos e atualmente sabe-se que no mínimo, 80% dos genes humanos apresentam variantes de splicing. A evolução dos Reguladores de Splicing Exônicos (ESRs) foi principalmente analisada através de abordagens filogenéticas. Embora esses estudos tenham revelado resultados consistentes (por exemplo, evidências de seleção negativa contra mudanças sinônimas que afetam ESRs) outros ainda parecem contraditórios, como a maior conservação filogenética e a maior taxa de evolução não-sinônima dos éxons alternativos em relação aos constitutivos. Nesta tese abordamos questões sobre o regime e a intensidade de seleção que atuam sobre os éxons e seus reguladores através da comparação da variação genética intra e inter-específica. Os resultados da tese demonstram que: 1. Há uma diferença na densidade e na intensidade de seleção negativa que atua sobre os ESRs de éxons constitutivos e alternativos. 2. Os inibidores de splicing tem papel principal na origem dos éxons alternativos a partir de éxons constitutivos e também nos casos de uso alternativo de sítios de splicing. 3. O nível de inclusão dos éxons está diretamente relacionado com a intensidade de seleção negativa sobre mudanças não-sinônimas / Splicing is the process by which introns are removed from a mRNA precursor and exons are ligated to form a mature mRNA. During this process several cis and trans factors are involved. Besides the canonical cis factors (e.g., splicing sites, branch point and polypyrimidine tract), Splicing Regulators - short sequences located in exons and introns - have an important role in assisting the spliceossome to correctly recognize exon/intron boundaries. Through alternative splicing, great transcript diversity is generated, and currently it is known that more than 80% of human genes present splicing variants. The evolution of Exonic Splicing Regulators (ESRs) has been mainly analyzed by interspecific comparisons. Although these studies have revealed consistent results (evidences of weak negative selection against synonymous variations that affect ESRs), other findings still appear inconsistent, for instance the reports on increased level of conservation and higher non-synonymous evolutionary rate in alternative than constitutive exons. The present thesis investigates the regime and intensity of natural selection on exons and their ESRs by comparing intra and interspecific genetic variation. We demonstrate that 1. ESRs from constitutive and alternative exons differ significantly in density and the intensity of negative selection. 2. The exonic splicing silencers have a major role in the origin of exons skipping from constitutive exons, and also on events of alternative usage of splicing sites. 3. There is a positive correlation between the exon inclusion level and the intensity of negative selection against non-synonymous variations

At the post-transcriptional level, the splicing of the pre-mRNA plays a vital role in cell fate determination and respond to biotic and abiotic stresses. Through alternative splicing, mRNAs variants can be produced from a single gene. SF3B is a heptameric protein complex that is essential for pre-mRNA splicing. It contains seven subunits: SF3b155, SF3b130, SF3b145, SF3b49, SF3b14b, P14/SF3b14a and SF3b10 and they play an important role in BS (branch point sequence) recognition. P14/SF3b14a interacts with the branch point Adenosine (BPA), directing the binding of U2 complex. Several studies performed on the mutations of SF3b complex as it is associated with many diseases. Further studies are needed to deeply analyze the molecular function of P14/SF3b14a in plant growth and development. CRISPR/Cas9 system employed in gene editing among eukaryotes. The capability of the system is not only limited to the scope of bioengineering but also for functional studies of genes. CRISPR/Cas9 system assists in revealing the function of genes and the genetic networks through establishing a functional knockout and can help in understanding the molecular basis behind these processes. Here, we report the successful targeted mutagenesis of SF3b14a/p14 gene in Oryza sativa and the recovery of homozygous and heterozygous mutants. Phenotypic analyses have shown that SF3b14a/p14 is hypersensitive to abiotic stresses compared to the wild type plants. Further physiological and molecular studies are needed to reveal the role of p14 during plant growth and development, and responses to abiotic stresses.

Plants acclimate to the changing environmental conditions by adjusting their molecular responses at different molecular levels including genome, epigenome, transcriptome, metabolome, and proteome levels to ensure survival. Plants adapt to abiotic stresses by establishing a ‘stress memory’ of previous exposures to mild stresses. Stress memory helps plants to develop tolerance and survive recurring exposures to the stress conditions. This memory establishes a new cellular state that differs from the state of unexposed naïve plants. This process is known as priming. Priming and the stress memory give the plants the possibility to acclimate to different biotic and abiotic stress conditions. The acquisition and maintenance of the stress-memory are two separate processes and crucial for successful tolerance to subsequent stress conditions. Priming promises to improve plant performance under severe stress conditions and enhance food production. Therefore, understanding the molecular basis of heat stress priming and stress-induced memory is of vital importance to plant biology. In this thesis, I investigated the role of transcriptional, post-transcriptional and metabolomic regulation controlling plant responses to heat stress, one of the major abiotic stresses affecting agriculture. I designed and established a heat stress priming strategy which reveals that heat stress-induced priming leads to the establishment of heat stress memory that permits plants to survive lethal temperatures. In this thesis, I analyzed the genome-wide differential gene expression, the alternative splicing patterns and regulation, and the reprogramming of the metabolic homeostasis that reprogram the establishment of the heat stress priming and stress-memory. I identified a set of candidate genes and metabolites playing key roles in the establishment of heat stress-induced memory. Intriguingly, it was possible also to establish a link between alternative splicing patterns and heat stress-induced memory. Subsequently, the knowledge of heat stress priming in Arabidopsis was translated into tomato crop plants, to improve their heat stress tolerance. This work enhances our understanding of the molecular basis of heat stress priming, and the establishment of heat stress memory, at transcriptional, post-transcriptional, and metabolomic levels. These findings can be translated into crop species to improve their survival under recurring heat stress conditions to improve world agriculture and food security.

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.