The SHAPE of U: Mapping Out Protective Elements in mRNA Escapees

A crucial step of the viral life cycle of Kaposi’s Sarcoma Herpesvirus (KSHV) lytic infection is the triggering of a massive RNA decay event termed “Host Shutoff”. Host Shutoff is driven by the viral endonuclease SOX which leads to the destruction of over 70% of the total transcriptome. This process cripples cellular gene expression and allows for viral reprograming of the cell for the purpose of viral replication. Co-evolution has led to the host developing a multitude of antiviral defenses aimed at preserving certain cellular RNAs linked to antiviral responses. One such defense are RNA secondary structures located within the 3’UTR of select host transcripts that protect them from SOX degradation. This structure, known as the SOX Resistant Element or SRE, has previously been isolated to a 200-nucleotide region found within the 3’UTR of the host transcript Interleukin-6. In this thesis, I sought to further define the structure of the IL-6 and other SREs using SHAPE-MaP to generate chemically-probed RNA structural models. Through this work, I demonstrated that the IL-6 SRE confers a form of active resistance to SOX cleavage, and based on structural analyses, likely acts as a scaffold for the recruitment of a protective ribonucleoprotein complex. This research highlights the importance of RNA secondary structures in influencing mRNA fate during viral infection and establishes the groundwork for understanding how these structural features can facilitate escape of cellular transcripts from viral endonucleases

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σS and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σS and, together, RprA and σS orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.Bildung und Forschung Project eBio:RNAsys BIO2013-44220-RMinisterio de Economía y Competitividad CSD2008-00013Junta de Andalucía CVI-587

Antiviral Immunity Directed by Small RNAs

Plants and invertebrates can protect themselves from viral infection through RNA silencing. This antiviral immunity involves production of virus-derived small interfering RNAs (viRNAs) and results in specific silencing of viruses by viRNA-guided effector complexes. The proteins required for viRNA production as well as several key downstream components of the antiviral immunity pathway have been identified in plants, flies, and worms. Meanwhile, viral mechanisms to suppress this small RNA-directed immunity by viruses are being elucidated, thereby illuminating an ongoing molecular arms race that likely impacts the evolution of both viral and host genomes

Human 5’-tailed Mirtrons are Processed by RNaseP

Approximately a thousand microRNAs (miRNAs) are documented from human cells. A third appear to transit non-canonical pathways that typically bypass processing by Drosha, the dedicated nuclear miRNA producing enzyme. The largest class of non-canonical miRNAs are mirtrons which eschew Drosha to mature through spliceosome activity. While mirtrons are found in several configurations, the vast majority of human mirtron species are 5’-tailed. For these mirtrons, a 3’ splice site defines the 3’ end of their hairpin precursor while a “tail” of variable length separates the 5’ base of the hairpin from the nearest splice site. How this tail is removed is not understood. Here we examine sequence motifs in 5’-tailed mirtrons and interactions with RNA turnover processes to characterize biogenesis processes. Through studying the high confidence 5’-tailed mirtron, hsa-miR-5010, we identify RNaseP as necessary and sufficient for “severing” the 5’ tail of this mirtron. Further, depletion of RNaseP activity globally decreased 5’-tailed mirtron expression implicating this endoribonuclease in biogenesis of the entire class. Moreover, as 5’-tailed mirtron biogenesis appears to be connected to tRNA processing we found a strong correlation between accumulation of tRNA fragments (tRFs) and 5’-tailed mirtron abundance. This suggests that dysregulation of tRNA processing seem in cancers also impact expression of the ~400 5’-tailed mirtrons encoded in the human genome

Post-Transcriptional Regulation Of The Eulkaryotic Transcriptome By The Covalent Rna Modicication N6-Methyladenosine

Post-Transcriptional regulation of the eukaryotic transcriptome by the covalent RNA modification N6-methyladenosine Stephen James Anderson Brian Gregory Once a messenger RNA molecule is transcribed, a myriad of RNA fate decisions must be made. How these fate decisions are made is often unclear, and elucidating factors determining these fate outcomes is an essential task in order to fully understand gene regulation. One poorly- understood but undoubtedly important factor in post-transcriptional gene regulation is the covalent modification of ribonucleotides. Much like DNA can have chemical groups added to a nucleotide within its primary sequence, RNA can be modified in a similar manner. These covalent modifications of RNA are a ubiquitous feature found within the RNA of all organisms. Dozens of these modifications have been described to date, yet the function or importance of most of these modifications remains unclear. One crucial RNA modification is N6-methyladenosine (m6A), as it is the most abundant known non-cap modification within the eukaryotic transcriptome. In this work, we characterize the role of m6A in the Arabidopsis transcriptome using various sequencing methods that demonstrate that m6A is an abundant mark that is largely maintained across differing Arabidopsis tissues and developmental stages. This prevalent mark promotes transcript stability in mNRAs involved in many important and diverse biological processes, such as salt stress. The absence of this mark results in endonucleolytic cleavage and degradation of the transcript in a highly specific and local manner. We further demonstrate that this modification modulates secondary structure throughout the transcriptome, and that m6A is associated with changes in RNA-binding protein association. Lastly, we turn our view to how an association between m6A and the m6A-specific binding protein YTHDC1 influences the development and transcriptome-wide splicing and polyadenylation pattern in the mouse germline. We demonstrate that in the absence of YTHDC1, widespread developmental, splicing, and polyadenylation defects occur, resulting in non-functional gametes. In total, this work greatly expands our knowledge and understanding of the biological importance and mechanisms of m6A-mediated post-transcriptional regulation

The Turnip mosaic virus and its effects on Arabidopsis thaliana gene expression

Utilizing natural and engineered viruses is an accepted approach to studying plant-virus interactions as it relates to symptomology. The majority of the research topics were generated by deciphering where short-comings in the literature existed. Specifically, how Turnip mosaic virus (TuMV) helper component protease (HC-Pro) small RNA (sRNA) binding affinity affects expression of genes correlated with disease phenotypes and studying debilitated viruses in a variety of RNA silencing deficient Arabidopsis thaliana plants. Taken as a whole, the research presented addressed the susceptibility of A. thaliana to TuMV. The first study was conducted to monitor genes implicated in symptomology in various RNA silencing pathway mutant backgrounds. I hypothesized that an in vitro approach, in conjunction with an in silico study would reveal the mechanism TuMV utilizes to regulate sRNA expression, post-infection. The second study focused on severe, moderate, and weak TuMV strains, versus A. thaliana response to pathogen challenge. I hypothesized that TuMV HC-Pro FRNK box mutants that differed in their ability to infect plants affected the function of host sRNA in graduated steps. I also postulated that these mutants might allow me to uncouple developmental abnormalities associated with disease progression and accumulation of the virus itself. In the final study, I combined my passion for plant pathology and molecular techniques to explore a topic unrelated to potyviruses. Conclusions based on analyzing the transcripts and sRNAs of genes correlated with TuMV disease symptomology, quantifying their expression in wild-type and RNA silencing defective A. thaliana plants, and characterizing various TuMV viruses lacking RNA silencing suppressor activity will be discussed. Future directions will also be introduced

DIFFERENT REPLICATION REQUIREMENTS IN THE HOMOLOGOUS 3' ENDS OF A POSITIVE STRAND RNA VIRUS AND ITS SUBVIRAL RNA

SatC is a noncoding subviral RNA associated with Turnip Crinkle Virus (TCV), a small (4054 nt) single-stranded (+)-strand RNA virus belonging to the Carmovirus genus. Because of its small size (356 nt) and TCV-derived 3' end, satC has been successfully used as a model to elucidate sequence and structural requirements for TCV RNA replication. Although satC is considered a model to identify cis-acting elements required for TCV replication, recent findings indicate distinct differences in structures and functions of these related sequences. RNA2D3D predicts that part of the TCV 3' end (H5, H4a, H4b and two pseudoknots) folds into an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits and is required for translation. SatC contains a similar 3' end with 6 nt differences in the 100 nt TSS region. RNA2D3D did not predict a similar structure for satC TSS region, and satC did not bind yeast ribosomes. satC nucleotides were changed into TCV TSS bases to determine which base differences are responsible for the loss of the TSS in satC. Changing these bases all increased ribosome binding but surprisingly none of them had an effect on satC accumulation in protoplasts and plants. Therefore satC may need these and other 3' end base differences for its required conformational switch for efficient replication, and not to inhibit ribosome binding. In vivo genetic selection (SELEX) of the linker sequence between H5 and the Pr showed the conservation of UCC, which led to the discovery of Ø2. Ø2 is required for both viral and satC accumulation in protoplasts. H5-Pr linker had no significant structural change after RdRp binding in satC, which is different with TCV H5-Pr linker. TCV H5-Pr linker had a major structural change upon RdRp binding, and is proposed to be involved in a conformational switch. Replacement of satC H4a with randomized sequence and scoring for fitness in plants by SELEX resulted in winning sequences that contain an H4a-like stem-loop. SELEX of H4a/H4b in satC generated two different structures: wt H4a/H4b-like structure and a single hairpin structure. Two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. With the protection of CP, satC can form higher amount of dimers that have additional nucleotides at the junction sites in the absence of TCV. The extra nucleotides are not necessarily associated with an active TCV RdRp

Basic Cell and Molecular Biology 5e: What We Know and How We Find Out

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