Yes / Alternative splicing of pre-mRNAs significantly contributes to the complexity of gene expression in higher organisms, but the regulation of the splice site selection remains incompletely understood. We have previously demonstrated that a chromatin-associated protein, AKAP95 (AKAP8), has a remarkable activity in enhancing chromatin transcription. In this study, we have shown that AKAP95 physically interacts with many factors involved in transcription and RNA processing, and functionally regulates pre-mRNA splicing. AKAP95 directly promotes splicing in vitro and the inclusion of a specific exon of an endogenous gene FAM126A. The N-terminal YG-rich domain of AKAP95 is important for its binding to RNA processing factors including selective groups of hnRNP proteins, and its zinc finger domains are critical for pre-mRNA binding. Genome-wide binding assays revealed that AKAP95 bound preferentially to proximal intronic regions on a large number of pre-mRNAs in human transcriptome, and AKAP95 depletion predominantly resulted in reduced inclusion of many exons. AKAP95 also selectively coordinates with hnRNP H/F and U proteins in regulating alternative splicing events. We have further shown that AKAP95 directly interacts with itself. Taken together, our results establish AKAP95 as a novel and mostly positive regulator of premRNA splicing and a possible integrator of transcription and splicing regulation, and support a model that AKAP95 facilitates the splice site communication by looping out introns through both RNA-binding and protein-protein interaction. / This work was supported by a UAB start-up fund to H.J.

Thesis (Ph.D.)--Virginia Commonwealth University, 2007. / Title from title-page of electronic thesis. Prepared for: Dept. of Biochemistry. Bibliography: leaves 125-142

No description available.

Gene expression in eukaryotes is dependent on the N-7 methylguanosine cap, located at the 5’ end of RNA pol II transcripts, which marks pre-mRNA for processing, stabilisation and translation initiation. The enzymes that catalyse the formation of the N-7 methylguanosine cap are recruited to RNA pol II at the initial stages of transcription. The final step in this process, N-7 methylation of the guanosine cap, is catalysed by the RNA guanine-7 methyltransferase, RNMT. RNA guanine-7 methylation is an essential process for cell viability and its up-regulation has been associated with cell transformation. However, the mechanistic details of RNMT function in mammalian cells remain elusive. In order to gain better understanding of the molecular mechanisms associated with RNA guanine-7 methylation, cellular RNMT complexes were purified from human cells and constituent proteins were identified using mass spectrometry. A novel component of the RNA guanine-7 methyltransferase complex was identified and designated as RAM (RNMT activating mini-protein). The vast majority of RNMT is found in a complex with RAM and vice versa.RAM is an RNA-binding protein, promoting recruitment of RNA to RNMT. RAM increases recombinant and cellular RNMT cap methyltransferase activity and is required for cap methylation in vivo. We therefore, describe RAM as an “obligate activator” of the human cap methyltransferase. As expected of a protein essential for cap methylation, RAM is required for gene expression, and RAM depletion results in loss of cell viability. Current studies are being focused on determining RAM/RNMT crystal structure as well as determining how the RNA guanine-7 methyltransferase complex is regulated within cells.

Frequent rearrangements, typically through homologous recombination in plant mitochondrial genomes often result in different upstream and downstream sequences for the same gene among a number of species. Transcription and RNA processing signals are therefore different, even among closely related plants. To evaluate the impact of DNA rearrangements on gene expression I conducted a comparative analysis of the S7 ribosomal protein gene (rps7) among a number of grasses: wheat, rice, maize, barley, rye, brome, Lolium and oats (grasses whose evolutionary divergence times range from about 5 to 60 Mya). Using circularized-RT-PCR to simultaneously map rps7 transcript termini I found that 3’ends for various RNA species are homogeneous, mapping to conserved sequences among plants. 5’ termini are more complex and can be both discrete and heterogeneous for different transcripts, both within and among plants. Genome rearrangements upstream of the rps7 start codon for some but not all species has led to plant-specific signals for both rps7 transcription and RNA processing. Termini for rps7 precursor species in wheat and Lolium are very discrete and likely use different upstream tRNAs as processing signals for end-cleavage. A number of potential stem-loop structures have also been identified at or near 5’ and 3’ termini which may function in maturation of transcript ends or provide transcript stability and protection from degradation by ribonucleases. C-to-U RNA editing of non-coding sequences, a rare event, was observed at multiple sites within the 5’ and 3’UTRs among plants. Some sites may even be developmentally regulated as CR-RT-PCR experiments were conducted using mitochondrial RNA isolated from seedlings and germinating embryos. Taken together, my observations demonstrate the frequency of upstream DNA rearrangements and the variety of signals used for expression of rps7 among grasses, providing new insights into the complexities of mRNA production in plant mitochondria.

Frequent rearrangements, typically through homologous recombination in plant mitochondrial genomes often result in different upstream and downstream sequences for the same gene among a number of species. Transcription and RNA processing signals are therefore different, even among closely related plants. To evaluate the impact of DNA rearrangements on gene expression I conducted a comparative analysis of the S7 ribosomal protein gene (rps7) among a number of grasses: wheat, rice, maize, barley, rye, brome, Lolium and oats (grasses whose evolutionary divergence times range from about 5 to 60 Mya). Using circularized-RT-PCR to simultaneously map rps7 transcript termini I found that 3’ends for various RNA species are homogeneous, mapping to conserved sequences among plants. 5’ termini are more complex and can be both discrete and heterogeneous for different transcripts, both within and among plants. Genome rearrangements upstream of the rps7 start codon for some but not all species has led to plant-specific signals for both rps7 transcription and RNA processing. Termini for rps7 precursor species in wheat and Lolium are very discrete and likely use different upstream tRNAs as processing signals for end-cleavage. A number of potential stem-loop structures have also been identified at or near 5’ and 3’ termini which may function in maturation of transcript ends or provide transcript stability and protection from degradation by ribonucleases. C-to-U RNA editing of non-coding sequences, a rare event, was observed at multiple sites within the 5’ and 3’UTRs among plants. Some sites may even be developmentally regulated as CR-RT-PCR experiments were conducted using mitochondrial RNA isolated from seedlings and germinating embryos. Taken together, my observations demonstrate the frequency of upstream DNA rearrangements and the variety of signals used for expression of rps7 among grasses, providing new insights into the complexities of mRNA production in plant mitochondria.

Frequent rearrangements, typically through homologous recombination in plant mitochondrial genomes often result in different upstream and downstream sequences for the same gene among a number of species. Transcription and RNA processing signals are therefore different, even among closely related plants. To evaluate the impact of DNA rearrangements on gene expression I conducted a comparative analysis of the S7 ribosomal protein gene (rps7) among a number of grasses: wheat, rice, maize, barley, rye, brome, Lolium and oats (grasses whose evolutionary divergence times range from about 5 to 60 Mya). Using circularized-RT-PCR to simultaneously map rps7 transcript termini I found that 3’ends for various RNA species are homogeneous, mapping to conserved sequences among plants. 5’ termini are more complex and can be both discrete and heterogeneous for different transcripts, both within and among plants. Genome rearrangements upstream of the rps7 start codon for some but not all species has led to plant-specific signals for both rps7 transcription and RNA processing. Termini for rps7 precursor species in wheat and Lolium are very discrete and likely use different upstream tRNAs as processing signals for end-cleavage. A number of potential stem-loop structures have also been identified at or near 5’ and 3’ termini which may function in maturation of transcript ends or provide transcript stability and protection from degradation by ribonucleases. C-to-U RNA editing of non-coding sequences, a rare event, was observed at multiple sites within the 5’ and 3’UTRs among plants. Some sites may even be developmentally regulated as CR-RT-PCR experiments were conducted using mitochondrial RNA isolated from seedlings and germinating embryos. Taken together, my observations demonstrate the frequency of upstream DNA rearrangements and the variety of signals used for expression of rps7 among grasses, providing new insights into the complexities of mRNA production in plant mitochondria.

Many cellular functions depend on the tightly regulated expression of various proteins. Canonical control of the protein expression is associated with transcriptional regulation. However, the small non-coding RNAs called microRNAs (miRNAs) were identified as post-transcriptional regulators of gene expression. In a typical manner, miRNAs originate similarly to the coding RNAs and are processed in a multi-step maturation process. It has been shown that miRNAs are very important for the proper functioning of tissues. Interestingly, the human nervous system contains over 70% of all miRNAs; thus, the maturation process has to be tightly regulated. However, despite the important role of miRNAs, little is known about the mechanisms regulating their biogenesis. In my PhD project, I showed that during early stages of neuronal differentiation, Lin28a controls levels of neuro-specific miRNA-9. I demonstrated that Lin28a binds to the conserved terminal loop (CTL) of pre-miRNA-9 and decreases the cellular levels of miRNA-9 during retinoic acid-mediated neuronal differentiation of mouse teratocarcinoma P19 cells. I revealed that the Lin28a-mediated inhibition of miRNA-9 production was uridylation-independent. Furthermore, constitutive expression of GFP-tagged Lin28a reduced the levels of let-7a but not miRNA-9, whereas untagged Lin28a inhibited both miR-9 and let-7a during the course of neuronal differentiation. Using small RNAseq analysis of P19 cells with constitutive expression of Lin28a I showed that it controls many more miRNAs than previously recognised. Intriguingly, many miRNAs were upregulated by Lin28a overexpression. I demonstrated with high-throughput, the limited function of GFP-tagged Lin28a results, and I also showed that untagged Lin28a inhibits the production of a number of brain-specific miRNAs including miRNA-9. Finally, I revealed that 3’-5’exoribonuclease Dis3l2 was responsible for uridylation-independent degradation of pre-miRNA-9. Altogether, my results provided evidence that Lin28a has both positive and negative roles in the regulation of miRNA production and has a dual role in triggering pre-miRNA degradation.

Frequent rearrangements, typically through homologous recombination in plant mitochondrial genomes often result in different upstream and downstream sequences for the same gene among a number of species. Transcription and RNA processing signals are therefore different, even among closely related plants. To evaluate the impact of DNA rearrangements on gene expression I conducted a comparative analysis of the S7 ribosomal protein gene (rps7) among a number of grasses: wheat, rice, maize, barley, rye, brome, Lolium and oats (grasses whose evolutionary divergence times range from about 5 to 60 Mya). Using circularized-RT-PCR to simultaneously map rps7 transcript termini I found that 3’ends for various RNA species are homogeneous, mapping to conserved sequences among plants. 5’ termini are more complex and can be both discrete and heterogeneous for different transcripts, both within and among plants. Genome rearrangements upstream of the rps7 start codon for some but not all species has led to plant-specific signals for both rps7 transcription and RNA processing. Termini for rps7 precursor species in wheat and Lolium are very discrete and likely use different upstream tRNAs as processing signals for end-cleavage. A number of potential stem-loop structures have also been identified at or near 5’ and 3’ termini which may function in maturation of transcript ends or provide transcript stability and protection from degradation by ribonucleases. C-to-U RNA editing of non-coding sequences, a rare event, was observed at multiple sites within the 5’ and 3’UTRs among plants. Some sites may even be developmentally regulated as CR-RT-PCR experiments were conducted using mitochondrial RNA isolated from seedlings and germinating embryos. Taken together, my observations demonstrate the frequency of upstream DNA rearrangements and the variety of signals used for expression of rps7 among grasses, providing new insights into the complexities of mRNA production in plant mitochondria.

Le CBC intervient dans de nombreuses étapes du métabolisme des ARN, telle que l’épissage, la maturation de l’extrémité 3’, la dégradation, l’export et la traduction. Ainsi, le CBC constitue un complexe majeur qui peut orchestrer les différentes étapes de maturation des ARN. Récemment, nous avons identifié le complexe CBCAP, composé de CBC, ARS2 et PHAX. Nous avons montré que la protéine ARS2 stimule la formation des extrémités 3’ de plusieurs familles d’ARN dont les snARN. De plus, ARS2 stimule le recrutement de PHAX sur le CBC. Ainsi, nous proposons un modèle où CBC-ARS2 stimule la formation de l’extrémité 3’ des pré-snARN et recrute PHAX pour favoriser leur export. Une autre étude a identifié un autre complexe le CBCN, constitué de CBC, Ars2, et de ZC3H18-NEXT au lieu de PHAX. CBCN recrute l’exosome et stimule la dégradation de certains ARN, comme les PROMPTS et les transcrits «read-through » des snARN et des ARNm d’histone. Ainsi, PHAX et ZC3H18 destinent leur ARN cibles vers l’export ou la dégradation. Il a été montré que PHAX reconnait et lie spécialement les ARN de petite taille. D’une manière remarquable, nos données de CLIP-Seq et de RIP suivie par des analyses avec des puces « All genes » montrent que PHAX lie aussi d’autres familles d’ARN. En effet, PHAX lie les ARNm ainsi que des ARN non-codant avec une légère préférence pour les snARN (en comparaison avec ZC3H18). Afin de mieux comprendre le rôle de PHAX et ZC3H18, j’ai tout d’abord démontré si les deux protéines se lient simultanément au CBC. Pour ce faire, J’ai réalisé des tests de compétitions entre PHAX et ZC3H18, in vivo, et j’ai montré que la surexpression de ZC3H18 déplace PHAX du CBC et vice versa. Puis en utilisant la technique de « Tethering Assays » j’ai pu montrer que PHAX et ZC3H18 ont des effets opposés sur la biogénèse des ARNm. De plus PHAX semble avoir un effet positif sur la maturation des ARNm et ce, en empêchant ZC3H18 et l’exosome d’être recruter. Nous avons aussi montré que la déplétion de PHAX et ZC3H18 a des conséquences fonctionnelles sur le taux des formes matures des snARN. Dans le but de caractériser la protéine ZC3H18, j’ai réalisé un crible double-hybride et j’ai montré que ZC3H18 interagit avec plusieurs facteurs d’épissage. J’ai aussi identifié les domaines de ZC3H18 impliqués dans ses différentes interactions. D’une manière intéressante, l’interaction de ZC3H18 avec certains facteurs d’épissage peut être exclusive à son interaction avec NEXT. De plus, des expériences de protéomique réalisés sur un des facteurs d’épissage trouvé dans le crible, montrent qu’il co-purifie au sein d’un complexe qui pourrait faire le lien entre la coiffe et la machinerie d’épissage. En accord avec ces résultats, nos données de RNA-seq montrent que la déplétion de ZC3H18 engendre un défaut d’épissage pour des introns qui sont proches de la coiffe et ceci pour un nombre restreint de gènes. Ainsi, notre travail décode davantage le rôle de la coiffe dans les différentes étapes de maturation des ARN et suggère un modèle où la séquence des transcrits naissant stimule la formation d’un complexe spécifique à cet ARN parmi plusieurs autres. / The cap binding complex (CBC) plays a key role in a number of gene expression pathways and has been proposed to participate in the discrimination of RNA families. It also enhances many RNA processing steps, including transcription, splicing, 3’end formation, degradation, export and translation.Recently, we identified the CBCAP complex, composed of CBC, Ars2 and PHAX. We showed that Ars2 stimulates snRNA 3'-end processing as well as PHAX binding to the CBC, hence coupling snRNA maturation with their export. Other studies showed that the CBC and ARS2 can form another complex that contains ZC3H18-NEXT instead of PHAX. This complex, named CBCN, is a cofactor of the RNA exosome and is involved in the degradation of cryptic RNAs such as PROMPTs and read-through transcripts at histone and snRNA genes. Thus, PHAX and ZC3H18 target specific families of capped RNA toward either export or degradation. Previous studies proposed that PHAX binds specifically to small RNAs and discriminates them over other RNA species. Surprisingly, our CLIP-Seq and RIP-microarrays data showed that in contrast to expectations, PHAX was not specific for snRNAs. It also binds mRNAs as well as other non-coding RNAs and has a weak preference for snRNAs comparing to ZC3H18. To better understand the role of PHAX and ZC3H18, Ifirst determined whether PHAX and ZC3H18 can bind simultaneously to the CBC. Competitive LUMIER IPs indicated that binding of these proteins is mutually exclusive. I then used tethering assays and could show that PHAX and ZC3H18 have opposite effect on mRNA biogenesis. These data go against a model where binding of PHAX or ZC3H18 discriminate RNA families, and instead suggest promiscuous binding for these proteins. In addition, PHAX may exert a positive effect on mRNA processing by preventing binding of ZC3H18 and recruitment of the RNA exosome. Last but not least, our RT-QPCR data show that PHAX and ZC3H18 depletions have functional consequences on the level of mature snRNA, and this is due to a competition between both proteins which occur on those snRNA read-through transcripts.To further explore the role of ZC3H18, I performed a two-hybrid screen and identified several splicing factors. I could validate these interactions, identify the domains involved and show that binding of some of these factors is exclusive with that of NEXT. Importantly, proteomic experiments with one of these factors identified a complex that makes the link between the cap and the splicing machinery. In agreement, RNA-Seq analysis of ZC3H18 knock-down cells showed alterations in splicing of cap-proximal introns, for a small set of genes.Altogether, this work reveals how the multiple roles of the RNA cap are achieved at the biochemical level, and suggests that the nascent RNA sequence triggers formation of one among several mutually exclusive complexes.