Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 217-230). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 138-170).

No description available.

No description available.

No description available.

As RNA helicases da família DEAD-box são enzimas que alteram as estruturas secundárias do RNA e auxiliam a formação de complexos ribonucleoproteicos, e são muito importantes em processos basais como a degradação dos RNAs e a biogênese dos ribossomos. A α-proteobactéria criotolerante Caulobacter crescentus é um modelo experimental interessante para compreender o papel destas enzimas em baixa temperatura. A caracterização fenotípica de linhagens mutantes simples de quatro RNA helicases DEAD-box permitiu demonstrar que a RNA helicase RhlE é necessária para o crescimento em baixa temperatura. Os mutantes duplos mostraram redução do crescimento à temperatura normal e em baixa temperatura, com perda de viabilidade e alterações morfológicas. Através de ensaios de complementação cruzada mostrou-se que seus papéis fisiológicos são até certo ponto redundantes. O mutante rhlE também apresentou redução na formação de biofilme. A medida da expressão relativa dos genes que codificam as RNA helicases mostrou um aumento na expressão de três destes genes em estresse frio, e a análise dos perfis ribossomais mostrou a possível participação destas três RNA helicases na biogênese do ribossomo. / The RNA helicases of the DEAD-box family are enzymes that modify RNA secondary structures and help the formation of ribonucleoprotein complexes, and are very important in basal processes such as RNA degradation and ribosome biogenesis. The cryotolerant α-proteobacterium Caulobacter crescentus is an interesting experimental model to understand the role of these enzymes at low temperature. The phenotypic characterization of strains with single mutations of four DEAD-box RNA helicases showed that RNA helicase RhlE is required for growth at low temperature. The double mutants showed reduction in growth at both normal and low temperatures, with loss of viability and morphological changes. Through cross-complementation assays it has been shown that their physiological roles are to some extent redundant. The rhlE mutant also showed reduction in biofilm formation. The relative expression of the genes encoding the RNA helicases showed an increase in the expression of three of these genes under cold stress, and the analysis of the ribosomal profiles showed the possible participation of these three RNA helicases in ribosome biogenesis.

RNA helicases are found in all kingdoms of life, functioning in all aspects of RNA biology mainly through modulating structures of RNA and ribonucleoprotein (RNP) complex. RNA structures have fundamental impacts on steps in gene expression, including transcription, pre-mRNA processing, and translation. However, the precise roles and regulatory mechanisms of RNA structures in co- and post-transcriptional processes remain elusive. By probing genome-wide RNA structures in vivo, a recent study suggested that ATP-dependent factors, such as RNA helicases, maintain the actively unfolded state of RNAs. Among all RNA helicases, DEAD-box proteins form the largest family in eukaryotes, and have been shown to remodel RNA/RNP structures both in vitro and in vivo. Nevertheless, for the majority of these enzymes, it is largely unclear what RNAs are targeted and where they modulate RNA/RNP structures to regulate co-transcriptional processes. To fill the gap, my research focused on identification of the RNAs and structures targeted by the DEAD-box protein Dbp2 in S. cerevisiae to uncover the cellular processes that Dbp2 is involved in.<br><div><div>My studies revealed a role of Dbp2 in transcriptional termination. Dbp2 binds to ~34% of yeast mRNAs and all snoRNAs, and loss of DBP2 leads to a termination defect as evidenced by RNA polymerase II (RNAPII) accumulation at 3’ ends of these genes. In addition, the binding pattern of Dbp2 in mRNAs is highly similar to Nrd1 and Nab3 in the Nrd1-Nab3-Sen1 (NNS) termination complex, and deletion of DBP2 leads to reduced recruitment of Nrd1 to its target genomic loci. In Dbp2 and NNS targeted 3’ UTRs, RNA structural changes resulted from DBP2 deletion also overlap polyadenylation elements and correlate with inefficient termination, and loss of stable structure in the 3’ UTR bypasses the requirement for Dbp2. These findings lead to a model that Dbp2 promotes efficient termination of transcription through RNA structure remodeling.</div><div>Interestingly, my research also revealed the requirement of DBP2 for efficient splicing, as loss of DBP2 leads to accumulation of unspliced pre-mRNAs. Moreover, this function is dependent on the helicase activity of Dbp2. Further studies are needed to characterize the molecular mechanism of how Dbp2 facilitates splicing in cells. Overall, my research demonstrated that DEAD-box RNA helicases remodel mRNA structure in vivo and that structural alteration can be essential for proper gene expression.</div></div>

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

Human immunodeficiency virus (HIV) is the causing agent of the acquired immune deficiency syndrome (AIDS). Like all retroviruses, HIV carries two copies of viral genomic RNA in each virion. HIV genome encodes three structural genes, including gag, pol and env, as well as two regulatory genes (rev and tat) and four accessory genes (vif, vpr, vpu and nef). It is noted that none of these nine viral proteins bears the helicase activity. Helicases are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). They are involved in every step of cellular RNA metabolisms. It is conceivable that HIV needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. / In this study, we found that a DEAD-box protein named DDX24 associates with HIV-1 Gag in an RNA-dependent manner but is not found within virus particles. Knockdown of DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.