Dodders (Cuscuta spp.) are parasitic plants that live by tapping into the vascular tissue of a host plant. Contents of the host phloem translocate readily into the parasite, and shared plasmodesmata have been documented between host cortical cells and dodder searching hyphae. Dodder is known to transmit viruses from one host to another, which is consistent with viral ability to traverse plasmodesmata (PD) with the aid of movement proteins (MPs). Plant endogenous mRNAs may also associate with specific proteins to pass through PD and traffic long distances in the phloem, a process that appears to play a role in coordination of development. We have evaluated the hypothesis that dodder is able to accumulate host phloem-mobile mRNAs by assaying lespedeza dodder (C. pentagona) for the presence of host transcripts. Reverse transcriptase PCR (RT-PCR) and tomato microarrays were used to probe RNA from dodder parasitizing tomato. Transcripts from four tomato genes were detected in dodder grown on tomato, but were not detected in control dodder grown on other hosts. Notable among these was LeGAI, a transcript previously shown to be phloem translocated. In addition, RT-PCR of RNA from dodder grown on pumpkin detected three mobile pumpkin mRNAs (CmNACP, CmSUTP1, and CmWRKYP). These results imply the existence of an extraordinary situation in which mobile mRNAs move from one plant into another, and raise questions about the role of this phenomenon in plant development and parasite pathogenicity. / Master of Science

Tesis por compendio / Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. The SUS1 gene of Saccharomyces cerevisiae is unusual, as it contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. In this thesis project, we have investigated whether the structure adopted by SUS1 RNA sequences contributes to regulate the splicing of this gene. Using in silico analyses together with NMR spectroscopy, gel electrophoresis and UV thermal denaturation experiments, we first show that the downstream intron (I2) of SUS1 forms a weakly-stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semi-quantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild-type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. The second part of the thesis project focuses on the exon located between the two introns of the SUS1 gene. This middle exon (E2) can be skipped during splicing, is generated in circular form, and has been found to influence the splicing of the flanking introns, an unusual situation in budding yeast where splicing mainly relies on intron recognition. Using NMR spectroscopy, gel electrophoresis, UV thermal denaturation and ribose 2'-OH modification experiments combined with computational predictions, we show that E2 of SUS1 comprises a conserved double-helical stem topped by a three-way junction. One of the hairpins emerging from the junction exhibited significant thermal stability and was closed by an unusually structured purine-rich loop. This loop contained two consecutive sheared G:A base pairs and was structurally related to the substrate loop of the VS ribozyme. Cellular assays revealed that three mutants containing altered E2 structures had impaired SUS1 expression and that a compensatory mutation restoring the conserved stem recovered expression to wild-type levels. Semi-quantitative RT-PCR experiments indicated that all mutants were capable of altering the quantities of unspliced and/or fully-spliced SUS1 RNA transcripts relative to wildtype. Overall, the results gathered in this thesis project indicate that RNA structures formed by the middle exon and the second intron of the S. cerevisiae SUS1 gene are relevant for splicing and also influence other processes of SUS1 mRNA biogenesis. / Sus1 es una proteína conservada implicada en remodelación de cromatina y biogénesis de moléculas de ARNm. El gen SUS1 de Saccharomyces cerevisiae es peculiar, ya que contiene dos intrones y sufre un proceso de ayuste (corte y empalme) alternativo, reteniendo uno o ambos intrones en respuesta a cambios en las condiciones ambientales. El ayuste del ARNpre-m de SUS1 puede permitir a la célula controlar la expresión de la proteína Sus1, pero los mecanismos que regulan este proceso son poco conocidos. En este proyecto de tesis hemos investigado si la estructura adoptada por secuencias de ARN de SUS1 contribuye a regular el proceso de ayuste de este gen. Utilizando análisis in silico junto con espectroscopia de RMN, electroforesis en gel y experimentos de desnaturalización térmica monitorizados por UV, primero demostramos que el ARN del segundo intrón (I2) del gen SUS1 forma una horquilla débilmente estable de 37 nucleótidos. Esta horquilla contiene nucleótidos del sitio de ramificación (branch site) en su bucle apical y nucleótidos del sitio 3' de empalme adyacentes al extremo inferior del tallo. A través de ensayos funcionales descubrimos que dos de cuatro mutantes que alteran la estructura de la horquilla I2 exhibían peor expresión de SUS1. Experimentos de RT-PCR semicuantitativos indicaron que todos los mutantes acumularon ARNpre-m SUS1 no ayustado y/o indujeron cambios en los niveles de ARNm maduro con respecto a la secuencia silvestre. Además, las funciones celulares de Sus1 relativas a desubicuitinación de histona H2B y transporte de ARNm se vieron afectadas en los mutantes de la horquilla I2 que inhibían el proceso de ayuste. La segunda parte de la memoria de tesis se centra en el análisis del exón central (E2) situado entre los dos intrones del gen SUS1. Este exón puede eliminarse durante el proceso de ayuste, se genera en forma circular, e influye en el procesamiento de los intrones adyacentes, una situación inusual para las regiones exónicas de S. cerevisiae, donde el ayuste se basa principalmente en el reconocimiento de intrones. Utilizando experimentos de espectroscopía de RMN, electroforesis en gel, desnaturalización térmica y modificación química combinados con predicciones computacionales, demostramos que el ARN del exón E2 de SUS1 forma un tallo conservado de doble hélice coronado por una intersección de tres hélices. Una de las horquillas que emergen de esta intersección presentó una estabilidad térmica significativa, así como un bucle apical rico en purinas inusualmente estructurado. Este bucle contiene dos pares de bases G:A consecutivos y está estructuralmente relacionado con el bucle de substrato de la ribozima VS. Ensayos celulares revelaron que tres mutantes con estructuras modificadas de E2 exhibían peor expresión de SUS1, y que una mutación compensatoria que restauraba el tallo conservado recuperaba la expresión a los niveles de la secuencia silvestre. Experimentos de RT-PCR semicuantitativos indicaron que todos los mutantes de E2 eran capaces de alterar las cantidades de transcritos ayustados y no ayustados de SUS1 con respecto a la secuencia silvestre. En general, los resultados obtenidos en este proyecto de tesis indican que las estructuras de ARN formadas por el exón central y el segundo intrón del gen SUS1 de S. cerevisiae son relevantes para el ayuste y otros procesos implicados en la biogénesis del ARNm del gen SUS1. / Sus1 és una proteïna conservada implicada a la remodelació de la cromatina i la biogènesi de l'ARNm. El gen SUS1 de Saccharomyces cerevisiae és inusual, ja que conté dos introns i s'empalma de manera alternativa, retenint un o ambdós introns en resposta a canvis en les condicions ambientals. L'empalmament de SUS1 pot permetre a la cèl·lula controlar l'expressió de Sus1, però els mecanismes que regulen aquest procés són segueixen sent desconeguts. En aquest projecte de tesi investiguem si l'estructura adoptada per seqüències d'ARN de SUS1 contribueix a regular l'empalmament d'aquest gen. Emprant anàlisi in silico juntament amb espectrometria de RMN, electroforesi en gel i experiments de desnaturalització tèrmica d'UV, es mostra primer que l'intró aigües a baix (I2) de SUS1 forma una estructura de forqueta de 37 nucleòtids feblement estable que conté el lloc de la branca a prop del seu bucle apical; i el lloc d'empalmamnet 3¿ després de l'extrem de la forqueta. Un assaig cel·lular va revelar que dos de quatre mutants que contenien estructures alterades de l'I2 havien modificat significativament l'expressió de SUS1. Els experiments semi-quantitatius de RT-PCR van indicar que tots els mutants acumulaven el pre-ARNm madur respecte al tipus salvatge. Concomitantment, les funcions cel·lulars de Sus1 a la desubiqüitinació de la histona H2B i l'exportació d'ARNm es van veure afectats als mutants de la forqueta d'I2 que inhibeixen l'empalmament. La segona part del projecte de tesi se centra a l'exó situat entre els dos introns del gen SUS1. Aquest exó (E2) es pot ometre durant l'empalmament, es genera amb forma circular, i s'ha trobat que influeix a l'empalmamet dels introns que flanquegen, una situació inusual al llevat on l'empalmament està basat principalment al reconeixement d'introns. Emprant espectroscòpia de RMN, electroforesi en gel, desnaturalització tèrmica d'UV i experiments de modificació de ribosa 2¿-OH combinats amb prediccions computacionals, mostrem que E2 de SUS1 comprén un tall conservat de doble hèlix corornat per una unió de tres vies. Una de les forquetes que emergeixen de la unió, va mostrar una estabilitat tèrmica significativa i va ser tapada per un bucle ric en purina inusualment estructurat. Aquest bucle contenia dos pars de bases G:A tallats consecutivament i estava estructuralment relacionat amb el bucle de substrat del ribozim VS. Els assajos cel·lulars van revelar que tres mutants que contenien estructures alterades de E2 havien alterat l'expressió de SUS1 i que una mutació compensatòria que restaurava el tall conservat recuperava l'expressió a nivells del tipus salvatge. Els assajos cel·lulars van revelat que tres mutants que contenien estructures alterades d'E2 havien alterat l'expressió de SUS1 i que una mutació compensatòria que restaurava el tall conservat recuperava l'expressió a nivell d'un tipus salvatge. Els experiments semi-quantitatius de RT-PCR van indicar que tots els mutants eren capaços d'alterar les quantitats de transcrits d'ARN de SUS1 no empalmats i/o empalmats en relació amb el tipus salvatge. En general, els resultats obtinguts en aquesta investigació indiquen que les estructures d'ARN formades per l'exó mitjà i el segon intró de SUS1 de S. cerevisiae són rellevants per l'empalmament i també influeixen a altres processos de biogènesi de l'ARN de SUS1 / Abuqattam, AN. (2017). IDENTIFICATION OF RNA STRUCTURES MODULATING THE EXPRESSION OF THE mRNA BIOGENESIS FACTOR SUS1 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90524 / Compendio

Nutrient absorption is important in all stages of life. As the diet of an animal changes from birth on, morphological and biochemical adaptation can be anticipated in order to accommodate changing demands. The main focus of the present study was to examine the relationship between age and diet on the potential for peptide transport via PepT1 in the gastrointestinal tract of lambs and to relate changes of peptide transport capability to morphological changes. A 2x4 factorial arrangement of treatments was used with 32 crossbred lambs. Four blocks were created based upon gender, birth type (single or twin), birth weight, and birth date. Lambs were randomly allotted at birth to receive or not to receive a creep diet. All lambs were allowed to nurse. Sampling times of 2, 4, 6, or 8 wk were randomly allotted to lambs. Samples for RNA extraction and histological evaluation were taken from the dorsal rumen, ventral rumen, omasum, duodenum, jejunum, and ileum. Villi were about 7% shorter (P < 0.09) in lambs receiving creep feed. Papillary height and width increased linearly (P < 0.001 and P < 0.0001, respectively) with age. Total and keratinized epithelial cells in the stomach decreased (P < 0.03 and P < 0.004, respectively) with age and were fewer (P < 0.0002 and P < 0.0001, respectively) in lambs receiving creep feed. Creep feeding appears to have slightly altered the mucosal structure of the small intestine and it was advantageous in that it stimulated papillary growth and thus predisposed the rumen for the introduction of feed into the diet. A 2.8 kb oPepT1 mRNA was present in all tissues studied by 2 wk, and age did not significantly influence the abundance of oPepT1 mRNA in the small intestine or stomach. In the small intestine, abundance of oPepT1 mRNA was greatest (P < 0.0007) in the jejunum. In the stomach, abundance of oPepT1 mRNA was greatest (P < 0.01) in the dorsal rumen. In the stomach, particularly in the rumen, a greater abundance of oPepT1 mRNA was observed in lambs not receiving the creep diet. It seems likely that a stimulus for development is coming from the non-luminal direction, possibly blood-borne, and may be involved in the ontogenesis of oPepT1. Peptide transport appears to be a physiologically important process in the young lamb and the rumen appears to be involved in the transport of peptides, particularly in nursing lambs. / Master of Science

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

In eukaryotes, mRNA decay pathways are important for cellular response to various physiological conditions and also function in co-translational quality control systems that target translationally aberrant mRNAs for degradation. My work on identification and characterization of novel components and pathways of mRNA degradation and quality control in Saccharomyces cerevisiae is summarized below.I have identified Edc3p as a novel protein important for mRNA decay. Deletion of Edc3p leads to a defect in mRNA decay in strains deficient in decapping enzymes and, in combination with a block to the 3' to 5' decay pathway, causes exaggerated growth defects and synthetic lethality. An Edc3p-GFP fusion protein localizes in processing bodies, which are specialized cytoplasmic foci containing decapping proteins. Together, these observations indicate that Edc3p directly interacts with the decapping complex to stimulate the mRNA decapping rate.Quality control during mRNA translation is critical for regulation of gene expression. My work shows that yeast mRNAs with defects in translation elongation, due to strong translational pauses, are recognized and targeted for degradation via an endonucleolytic cleavage in a novel process referred to as No-Go Decay (NGD). The cellular mRNA decay machinery degrades the 5' and 3' cleavage products produced by NGD. NGD is translation-dependent, occurs in a range of mRNAs and can be induced by a variety of elongation pauses. These results indicate NGD may occur at some rate in response to any stalled ribosome.I also show that two highly conserved proteins, Dom34p and Hbs1p, homologous to the eukaryotic release factors eRF1 and eRF3 respectively, are required for NGD. Further characterization of the No-Go decay pathway indicates that Dom34p function during NGD is conserved across species. Identification of RPS30, a small ribosomal protein as a trans-acting factor during NGD suggests that the ribosome may have a novel role during NGD. Other experiments indicate that the No-Go decay pathway may cross talk with the unfolded protein response pathway. The identification of No-Go decay as a novel quality control pathway during translation elongation supports the existence of a global cellular mechanism for maintenance of translational quality control.

Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression. First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle.  Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre-mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay. Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability. Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression. In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.

The splicing of pre-mRNA transcripts is catalyzed by a huge and dynamic machinery called spliceosome. The spliceosomal complex consists of five small nuclear ribonucleoprotein (snRNP) particles and hundreds of non-snRNP proteins. Biogenesis of spliceosomal snRNPs is a multi-step process, the final steps of which take place in a specialized sub-nuclear compartment, the Cajal body. However, molecular details of snRNP targeting to the Cajal body remain mostly unclear. Our previous results revealed that SART3 protein is important for accumulation of U4, U5 and U6 snRNPs in Cajal bodies, but how SART3 binds snRNP particles is elusive. SART3 has been identified as a U6 snRNP interaction partner and U4/U6 di-snRNP assembly factor. Here, we show that SART3 interacts with U2 snRNP as well, and that it binds specifically immature U2 particles. Next, we provide evidence that SART3 associates with U2 snRNP via Sm proteins, which are components of the stable snRNP core and are present in four out of five major snRNPs (i.e. in U1, U2, U4 and U5). We propose that the interaction between SART3 and Sm proteins represents a general SART3-snRNP binding mechanism, how SART3 recognizes immature snRNPs and quality controls the snRNP assembly process in Cajal bodies.

Pre-mRNA splicing is a vital process of gene expression important for all eukaryotic organisms. For the proper function of this very complex and dynamic event the presence of few specialized RNA and many proteins that hold a variety of tasks is necessary, not only inside the splicing complex itself, but also beyond this complex. The Prp45 is one of the proteins involved in pre-mRNA splicing in yeast Saccharomyces cerevisiae. Its human homologue, SNW1/SKIP, is involved in splicing but also in other crucial cell processes. The Prp45 protein was reliably reported only to participate in the second transesterification reaction of splicing. But there are also data suggesting its possible involvement in the first transesterification reaction. This work provides further evidences linking protein Prp45 with the first splicing reaction, obtained by the research of cells carrying the mutant allele prp45(1-169). Cells carrying this allele show dropped splicing and accumulation of pre-mRNAs. This thesis therefore also investigated the possible influence of Prp45 protein on the RNA export from the nucleus to the cytoplasm. But no connection between this protein and RNA transport was discovered. Keywords pre-mRNA splicing; Saccharomyces cerevisiae; Prp45; Mer1; Mud2; Prp22; Rrp6; AMA1; SNW1/SKIP

In eukaryotes, translation initiation is guided by up to twelve protein initiation factors (eIFs) and begins with the formation of the 43S pre-initiation complex (PIC) composed of the small ribosomal subunit (40S), eIF2.GTP/Met-tRNAi Met ternary complex, and eIFs 1, 1A, 3 and 5. The 43S PIC subsequently interacts with the 5'end of an mRNA (an mRNA recruitment step) and thus formed 48S PIC travels in 5' to 3' direction along the mRNA leader sequence to locate the AUG start codon (this presumably linear movement is generally known as scanning). Start site selection results in the dissociation of the initiation factors and joining of the large (60S) ribosomal subunit to form the 80S initiation complex poised for elongation. Eukaryotic initiation factor 3 (eIF3) plays a critical role in most of these events; however, the molecular details of most of its contributions are still unknown to us. Previous in vivo studies generated numerous mutations in all eIF3 subunits with specific defects either in the PICs assembly or in the following steps such as scanning, AUG recognition, etc. To understand the exact role of eIF3 in this intriguing process at the molecular level, we have embarked on a study that aims to dissect the individual functions of each eIF3 subunit in translation initiation using the purified...

hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...