Elucidating the roles of PARP1 and RBBP6 in the regulation of pre-mRNA 3' end processing

The mature 3' ends of most mRNAs are created by a two-step reaction that involves an endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream product. The 3' processing machinery is composed of four multisubunit complexes, which, together with a few other proteins, constitute the core components required for cleavage and polyadenylation. A proteomic analysis led to the identification of approximately 80 proteins that associate with the human pre-mRNA 3' processing complex, including new core 3' factors and other proteins that might mediate crosstalk between 3' processing and other nuclear pathways. This thesis focuses on two of the newly identified proteins, which we found particularly intriguing: PARP1 and RBBP6. PARP1 is an enzyme that, when activated, catalyzes the polymerization of ADP-ribose units from donor NAD molecules to acceptor proteins, a reaction known as PARylation. This post-translational modification has been shown to modulate critical events such as DNA damage response and transcription. We found that PARP1 binds PAP, the enzyme responsible for polyadenylating the 3' ends of mRNAs, and modifies it by PARylation. In vivo PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. Finally, we show that the observed inhibition reflects decreased PAP association with 3' end of genes. These results identify PARP1 as a regulator of polyadenylation during thermal stress and show for the first time that PARylation can control gene expression by modulating processing of mRNA. The second project involves RBBP6, a large multidomain protein that is known to interact with p53 and Rb. The N-terminal part of the human RBBP6 includes a DWNN domain, which is particularly interesting because it adopts a ubiquitin-like fold and, in addition to forming part of the full-length RBBP6 protein, is also expressed as a small protein (RBBP6 isoform3) which has been shown to be downregulated in several human cancers. We found that RBBP6 is essential for the cleavage activity of the 3' processing complex and that an N-terminal derivative of RBBP6 (RBBP6-N), containing only the DWNN, Zinc and Ring domains, is enough to rescue cleavage activity. The RBBP6 and RBBP6 isoform3 can compete with each other in binding to Cstf64 (an interaction mediated by the DWNN domain). In addition, overexpression of isoform3 inhibits cleavage raising intriguing possibilities of modulation of 3' processing by fine-tuning the levels of the two RBBP6 isoforms. To better characterize the function of RBBP6 globally, we also performed genome-wide analysis, both by microarray and deep sequencing. Following RBBP6 knockdown we observed a general lengthening of 3' UTRs accompanied by an overall downregulation in gene expression, especially of RNAs with AU-rich 3'UTRs. We show that this is the result of a defect in their 3' cleavage and subsequent degradation by the exosome. All together our results point to a role for RBBP6 as a new core 3' processing factor able to regulate the expression of AU-rich mRNAs

-Dual nucleoside therapy for HIV infection: analysis of results and factors influencing viral response and long term efficacy.

We performed a retrospective analysis of our experience with dual nucleoside regimens to look for predictors of long term benefit. We evaluated a cohort of 68 HIV-infected patients treated at 3 Italian hospital-based facilities. The results were analysed using univariate and multivariate statistical analyses. Fourty-three males and 25 females were treated for 22 ± 14 months. Sixty three patients (92.6%) suffered no or low-grade side-effects. Thirty-four patients (50 %) reached a viral load 150/μl pre-treatment viremia 1,500/μl, and no previous exposure to NRTI. Total lymphocyte counts and CD4+ T-cells showed a significant correlation. Dual NRTI regimens may be still considered for patients unable to tolerate HAART regimens and presenting with favourable predictors of response

The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system

The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAVdependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation

Epigenetic control of alternative mRNA processing at the imprinted Herc3/Nap1l5 locus

Alternative polyadenylation increases transcriptome diversity by generating multiple transcript isoforms from a single gene. It is thought that this process can be subject to epigenetic regulation, but few specific examples of this have been reported. We previously showed that the Mcts2/H13 locus is subject to genomic imprinting and that alternative polyadenylation of H13 transcripts occurs in an allele-specific manner, regulated by epigenetic mechanisms. Here, we demonstrate that allele-specific polyadenylation occurs at another im-printed locus with similar features. Nap1l5 is a retrogene expressed from the paternally inherited allele, is situated within an intron of a ‘host ’ gene Herc3, and overlaps a CpG island that is differen-tially methylated between the parental alleles. In mouse brain, internal Herc3 polyadenylation sites upstream of Nap1l5 are used on the pater-nally derived chromosome, from which Nap1l5 is expressed, whereas a downstream site is used more frequently on the maternally derived chromo-some. Ablating DNA methylation on the maternal allele at the Nap1l5 promoter increases the use of an internal Herc3 polyadenylation site and alters exon splicing. These changes demonstrate the influ-ence of epigenetic mechanisms in regulating Herc3 alternative mRNA processing. Internal Herc3 polyadenylation correlates with expression levels of Nap1l5, suggesting a possible role for transcrip-tional interference. Similar mechanisms may regulate alternative polyadenylation elsewhere in the genome

Molecular regulation of alternative polyadenylation (APA) within the Drosophila nervous system

Alternative polyadenylation (APA) is a widespread gene regulatory mechanism that generates mRNAs with different 3′-ends, allowing them to interact with different sets of RNA regulators such as microRNAs and RNA-binding proteins. Recent studies have shown that during development, neural tissues produce mRNAs with particularly long 3′UTRs, suggesting that such extensions might be important for neural development and function. Despite this, the mechanisms underlying neural APA are not well understood. Here, we investigate this problem within the Drosophila nervous system, focusing on the roles played by general cleavage and polyadenylation factors (CPA factors). In particular, we examine the model that modulations in CPA factor concentration may affect APA during development. For this, we first analyse the expression of the Drosophila orthologues of all mammalian CPA factors and note that their expression decreases during embryogenesis. In contrast to this global developmental decrease in CPA factor expression, we see that cleavage factor I (CFI) expression is actually elevated in the late embryonic central nervous system, suggesting that CFI might play a special role in neural tissues. To test this, we use the UAS/Gal4 system to deplete CFI proteins from neural tissue and observe that in this condition, multiple genes switch their APA patterns, demonstrating a role of CFI in APA control during Drosophila neural development. Furthermore, analysis of genes with 3′UTR extensions of different length leads us to suggest a novel relation between 3′UTR length and sensitivity to CPA factor expression. Our work thus contributes to the understanding of the mechanisms of APA control within the developing central nervous system

Multilayer regulatory mechanisms control cleavage factor I proteins in filamentous fungi

Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs

Reprogramming the assembly of unmodified DNA with a small molecule

The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA ‘alphabet’ by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials

Genetic determinants in a critical domain of ns5a correlate with hepatocellular carcinoma in cirrhotic patients infected with hcv genotype 1b

HCV is an important cause of hepatocellular carcinoma (HCC). HCV NS5A domain‐1 interacts with cellular proteins inducing pro‐oncogenic pathways. Thus, we explore genetic variations in NS5A domain‐1 and their association with HCC, by analyzing 188 NS5A sequences from HCV genotype‐1b infected DAA‐naïve cirrhotic patients: 34 with HCC and 154 without HCC. Specific NS5A mutations significantly correlate with HCC: S3T (8.8% vs. 1.3%, p = 0.01), T122M (8.8% vs. 0.0%, p < 0.001), M133I (20.6% vs. 3.9%, p < 0.001), and Q181E (11.8% vs. 0.6%, p < 0.001). By multivariable analysis, the presence of >1 of them independently correlates with HCC (OR (95%CI): 21.8 (5.7–82.3); p < 0.001). Focusing on HCC‐group, the presence of these mutations correlates with higher viremia (median (IQR): 5.7 (5.4–6.2) log IU/mL vs. 5.3 (4.4–5.6) log IU/mL, p = 0.02) and lower ALT (35 (30–71) vs. 83 (48–108) U/L, p = 0.004), suggesting a role in enhancing viral fitness without affecting necroinflammation. Notably, these mutations reside in NS5A regions known to interact with cellular proteins crucial for cell‐cycle regulation (p53, p85‐PIK3, and β‐ catenin), and introduce additional phosphorylation sites, a phenomenon known to ameliorate NS5A interaction with cellular proteins. Overall, these results provide a focus for further investigations on molecular bases of HCV‐mediated oncogenesis. The role of these NS5A domain‐1 mutations in triggering pro‐oncogenic stimuli that can persist also despite achievement of sustained virological response deserves further investigation

Specificity factors in cytoplasmic polyadenylation

Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm is called cytoplasmic polyadenylation. It was first discovered in oocytes and embryos, where it has roles in meiosis and development. In recent years, however, has been implicated in many other processes, including synaptic plasticity and mitosis. This review aims to introduce cytoplasmic polyadenylation with an emphasis on the factors and elements mediating this process for different mRNAs and in different animal species. We will discuss the RNA sequence elements mediating cytoplasmic polyadenylation in the 3′ untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In addition to describing the role of general polyadenylation factors, we discuss the specific RNA binding protein families associated with cytoplasmic polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4), Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins (ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C (BICC1). Some emerging themes in cytoplasmic polyadenylation will be highlighted. To facilitate understanding for those working in different organisms and fields, particularly those who are analyzing high throughput data, HUGO gene nomenclature for the human orthologs is used throughout. Where human orthologs have not been clearly identified, reference is made to protein families identified in man