The functions of the RNA polymerase II CTD in transcription and RNA processing

RNA polymerase II (RNAP II), transcribing messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), and non-coding RNAs (ncRNAs), is composed of 12 subunits. Rpb1, the largest subunit with catalytic polymerase activity, possesses a unique c-terminal domain (CTD) that consists of tandem heptad repeats with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). Somewhat reflecting the complexity of the organism, the number of repeats varies, from 26 in yeast to 52 in vertebrates. The CTD, intensively phosphorylated during transcription, serves a means to coordinate transcription and RNA processing- capping, splicing, and 3' end formation. For example, Ser 5, phosphorylated in the start of transcription, promotes the recruitment of capping enzyme, and Ser 2 phosphorylation facilitates RNA 3' end formation and transcription termination by acting as a landing pad for Pcf11. Detailed introduction is described in Chapter 1. Because of the importance of the CTD in these events, I created an Rpb1 conditional knock-out DT40 cell line (DT40-Rpb1) to further study the CTD with an initial focus on Thr 4, the function of which was unclear. Using DT40-Rpb1 system, we found that Thr 4 was phosphorylated in yeast, fly, chicken, and human cells, and cyclin-dependent kinase (CDK9) was likely the kinase to carry out this phosphorylation. We further provide evidence that Thr 4 functions in histone mRNA 3' end formation (presented mostly in chapter 2 of this thesis). Chapter 3 mainly describes the studies regarding Ser 2, Ser 5, and Ser 7. Based on the DT40-Rpb1 cell line, I created stable cell lines expressing an Rpb1 carrying a CTD with Ser 2, Ser 5, or Ser 7 mutated to alanine, and investigated the phenotypes of these cells. We found that cells expressing an Rpb1 with S2A or S5A mutation were defective in transcription and RNA processing. Contrary to previous findings, we found Ser 7 was not involved in snRNA expression and 3' end processing. In fact, no phenotypes associated with Ser 7 mutation were detected by our measurements. Extending previous Thr 4 studies, we showed in vitro and in vivo that Fcp1 dephosphorylated Thr 4. Finally, Chapter 4 describes what we have found the functions of CTD Tyr 1. Using the DT40-Rpb1 cells, I created stable cell lines expressing an Rpb1 with all Tyr residues mutated to phenylalanine (Phe). We found these cells were inviable, and the mutant Rpb1-Y1F was degraded to a CTD-less protein. Interestingly, the instability of Rpb1-Y1F was restored by reintroduction of one Tyr residue at the last heptad repeat. Further analysis provided evidence showing the involvement of Tyr phosphorylation in preventing Rpb1 from degradation by the 20S proteasome. Next, using ChIP assay, we showed Tyr phosphorylation was detected mostly at promoters, indicating a function of Tyr phosphorylation in transcription initiation. Indeed, transcription initiation defects were uncovered by assessing the recruitment of general transcription factors in cells with Y1F mutation. Extending this, we found an accumulation of upstream antisense RNAs in about one hundred reference genes by RNA-Seq analysis

The Musashi RNA Binding Proteins Are Regulators of Alternative Splicing and Protein Expression in Photoreceptor Cells

The Musashi (Msi) family of RNA binding proteins consists of two paralogs, Msi1 and Msi2, that are highly conserved across species. The two paralogs have emerged as factors that promote stem cell proliferation by post-transcriptionally regulating translation. In addition to their expression in stem cells, the Musashi proteins are also expressed in postmitotic neurons, including the photoreceptor cells. The Musashi proteins have been observed to maintain high expression levels in the postmitotic photoreceptors within the eye of both invertebrates and vertebrates. These observations suggest an additional role in the maintenance of terminally differentiated neurons. Building upon these observations, we investigated the role of Musashi individually and in combination in mature photoreceptors. Using a tamoxifen-inducible mouse model, I generated single and combined deletion of Msi1 and Msi2 in mature photoreceptor cells. Our results show that the Musashi proteins are required for the function and viability of mature photoreceptors. Global analysis of the Msi1 targets in the retina showed binding to UAG motifs predominantly located in introns and 3’-UTRs. Using RNA-sequencing and proteomics analysis, with the incorporation of the publicly available single-cell RNA seq, we found that in mature photoreceptors, the Musashi enhance the expression of proteins in high demand. Among these targets are proteins needed for the daily regeneration of the light sensory organelle of the photoreceptors. Collectively, the data provide new insights on the targets, possible molecular mechanisms, and function of the Musashi in mature photoreceptors. The results support a model of the Musashi proteins acting as a posttranscriptional activator for protein expression in mature photoreceptors. In the course of our work, an unusual behavior of the 13A4 antibody to prominin-1 (Prom1) prompted us to analyze its epitope. Prom1 is a transmembrane protein with a role in the morphogenesis of photoreceptor outer segment disk membranes. Mutations in the Prom1 gene have resulted in various forms of retinal degeneration affecting rods and cones. Scanning deletion mutagenesis and structural modeling demonstrated that mAB 13A4 recognizes a structural epitope that is affected by the inclusion of the alternative exon 19 during photoreceptor maturation. Consequently, the reactivity of mAB 13A4 towards the photoreceptor specific isoform of PROM1 is significantly reduced on a Western blot leading to gross underestimation of PROM1 protein levels in the retina

Identification of Key Molecules Involved in the Protection of Vultures Against Pathogens and Toxins

This is an open access article distributed under the terms of the Creative Commons Attribution License.This work was supported by the Junta de Comunidades de Castilla-La Mancha (JCCM), project PII1I09-0243-4350.Peer Reviewe

Mechanisms of action and co-optive evolution for hypervariable courtship pheromones in plethodontid salamanders.

Pheromones are an important type of chemical cue used by most animals to convey information between individuals. For more than 100 million years, male plethodontid salamanders have utilized a system of non-volatile, proteinaceous pheromones to regulate female mating behavior and receptivity. One of these pheromone components, Plethodontid Modulating Factor (PMF), is a hypervariable protein related to the three-finger protein (TFP) superfamily. Previous studies revealed that PMF persists as a rapidly evolving multi-isoform mixture. However, many characteristics of PMF as a pheromone remained undetermined, including gene structure and transcriptional regulation, translational regulation, protein structure, evolutionary mechanisms, and the isoform effects on female behavior and neurophysiology. Therefore, the broad aim of this dissertation was to characterize the mechanisms of action and evolution for PMF using the red-legged salamander, Plethodon shermani. The molecular and proteomic diversity of PMF was determined by RT-PCR and mass spectroscopy. The PMF complex is the product of at least 13 gene duplications in 3 gene classes containing highly conserved 5’ and 3’ untranslated regions (UTRs). These UTRs are bound by cold inducible RNA binding protein, which likely plays a key role in coordinating expression of the many diverse PMF isoforms during gland development. Using mass spectroscopy and multidimensional NMR, the 3D structure of the most abundant PMF isoform was determined to have a novel structure compared to all other TFPs. In particular, an altered disulfide bonding pattern promoted greater backbone flexibility in the most rapidly evolving segments of PMF to possibly enhance male pheromone and female receptor interactions. Functional assays testing different mixtures of PMF isoforms revealed that isoform diversity is a key requirement for increasing female receptivity, likely through synergistic interactions in the vomeronasal organ and/or the brain. Examination of pheromones in a different plethodontid species (P. cinereus) revealed that the majority of PMF duplications occurred within the last ~20 million years. In summary, in response to female sexual selection, the PMF gene complex has evolved through an unusual disjunctive evolutionary process as part of a birth-and-death model of gene evolution to permit coordinated expression of dozens of flexible proteins that synergistically function to regulate female behavior

Compensatory Relationship Between Exonic Splicing Enhancer, Splice Site and Protein Function

The process of pre-mRNA splicing involves the removal of intronic sequences from the pre-mRNA and it is directed by intronic cis acting elements know as the 5’ and 3’ splice sites that mark the boundaries of the exons. Over the two decades, however, it has become clear that exons encode for auxiliary splicing signals that either enhance or perturb their inclusion in the final mRNA product. It is possible that the evolution of mRNA sequences could be conditioned by the presence of these exonic cis-acting splicing regulatory elements and not mainly by the selection of optimal protein function. To explore this hypothesis, I have investigated how the need for ESE influences the gene evolution of a paralogous gene family, specifically the human Alkaline Phosphatases (ALPs). In this work, I have identified in correspondence to a weak 3’splice site, two ESE sequences in the placental ALP exon 4, and demonstrate that the ESE are necessary for the exon inclusion in the mRNA due to the weak 3’splice sites. Furthermore, I show that they are absent in the corresponding exon of the non-tissue specific ALP transcript, specifically exon 5 that carries a strong 3’ splice site. Most importantly, the localization of the ESEs correspond to an area that in the paralogous non-tissue specific ALP gene differs in amino acid composition with respect, not only to the placental ALP where I mapped the ESEs but also to the other members of the family, where this area is well conserved. These amino acid changes may represent a possible evolutionary constraint on enzymatic activity, in keeping with this hypothesis, substituting the amino acids in the region of the ESE for those of the paralogous non-tissue specific ALP gene increases the enzymatic activity. Thus splicing-related constraints challenge the primacy of biochemical function in rates of protein evolution

CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION

The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar

STRAIN-SPECIFIC PROTEIN INTERACTION AND LOCALIZATION OF TWO STRAINS OF POTATO YELLOW DWARF VIRUS AND FUNCTIONAL DOMAINS OF THEIR MATRIX PROTEIN

Potato yellow dwarf virus (PYDV) is the type species of the genus nucleorhabdovirus which is typified by its nucleotropic characters of the members. The virus accomplishes its replication and morphogenesis in the nuclei of infected cells. Two strains, Constricta strain (CYDV) and Sanguinolenta strain (SYDV) have been described at the level of vector-specificity. CYDV is vectored by Agallia constricta and SYDV is transmitted by Aceratagllia sanguinolenta. The full-length genome of CYDV was sequenced. The 12,792 nt antisense genome encodes seven open reading frames in the order of, nucleocapsid protein (N), unknown protein (X), phosphoprotein (P), movement protein (Y), matrix protein (M), glycoprotein (G), and large polymerase protein (L). The features of each protein including a nuclear localization signal, isoelectric point, and transmembrane domain, were determined by predictive algorithms. The gene coding region was flanked by leader and trailer, and each ORF was separated by a conserved intergenic junction. In the intergenic junctions, the highly conserved cis-regulatory elements, polyadenylation signal, gene spacer, and transcription start site, were identified. The similarities of amino acid sequences between each cognate protein of SYDV and CYDV were higher than 80% except for X and P proteins. The protein localization and interaction assays of each CYDV protein identified strain-specific associations in comparison with those of SYDV and generated unique protein interaction and localization map compared to SYDV. Phylogenetic analysis using L protein identified that CYDV forms a clade with other leafhopper-transmitted rhabdoviruses. Protein sequence comparisons revealed that CYDV X has greater similarity to the cognate protein of Eggplant mottle disease virus than to SYDV X. The localization patterns of CYDV-N and -Y were different compared the cognate proteins of SYDV. The functional nuclear export domain of SYDV M was identified using c-terminal fragments of the Mwt(aa 211-243), MLL223AA(aa 211-243), and MKR225AA(aa 211-243). Based on the data, the functional domains M mediating membrane association, nuclear import and export were mapped for both strains and suggested a model whereby M mediates intra- and intercellular movement of PYDV nucleocapsid