Cells infected with herpes simplex virus show a rapid cessation of protein synthesis and a dramatic decline in the levels of mRNA; a process known as host shutoff. This effect is attributed to a viral tegument protein called the virion host shutoff protein, or vhs. The mechanism by which vhs induces mRNA degradation is not yet understood. It is not known whether vhs possesses RNase activities or if it acts in combination with other cellular factors. To gain a better understanding of the function of vhs, I examined RNA degradation in detail by analyzing the RNA decay products generated in the presence of vhs. 𝘐𝘯 𝘷𝘪𝘷𝘰 experiments, performed by infecting murine erythroid leukemia cells with HSV, revealed that beta-globin mRNA is rapidly degraded, in the presence of vhs, without being converted to detectable decay intermediates. The half-life of this mRNA was 15 and 60 minutes for HSV-2 and HSV-1, respectively. Using vhs translated in a rabbit reticulocyte lysate system, I found that vhs induced rapid decay at the 5' end of a capped RNA molecule. The decay event was endonucleolytic and occurred at preferred sites downstream of the cap, generating capped oligonucleotides. Unlike influenza RNA polymerase, the cleavage event did not occur at a fixed distance from the cap since capped oligos of differing size were generated from different RNA substrates. My data indicate that vhs induced cleavage exhibits a strong, but not absolute preference for RNAs possessing an m⁷G cap, which may account for vhs' specificity for m RNAs 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)

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

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...