Force for ancient and recent life: viral and stem-loop RNA consortia promote life.

Lytic viruses were thought to kill the most numerous host (i.e., kill the winner). But persisting viruses/defectives can also protect against viruses, especially in a ubiquitous virosphere. In 1991, Yarmolinsky et al. discovered the addiction modules of P1 phage, in which opposing toxic and protective functions stabilize persistence. Subsequently, I proposed that lytic and persisting cryptic virus also provide addiction modules that promote group identity. In eukaryotes (and the RNA world), a distinct RNA virus-host relationship exists. Retrovirurses/retroposons are major contributors to eukaryotic genomes. Eukaryotic complexity appears to be mostly mediated by regulatory complexity involving noncoding retroposon-derived RNA. RNA viruses evolve via quasispecies, which contain cooperating, minority, and even opposing RNA types. Quasispecies can also demonstrate group preclusion (e.g., hepatitis C). Stem-loop RNA domains are found in long terminal repeats (and viral RNA) and mediate viral regulation/identity. Thus, stem-loop RNAs may be ancestral regulators. I consider the RNA (ribozyme) world scenario from the perspective of addiction modules and cooperating quasispecies (i.e., subfunctional agents that establish group identity). Such an RNA collective resembles a "gang" but requires the simultaneous emergence of endonuclease, ligase, cooperative catalysis, group identity, and history markers (RNA). I call such a collective a gangen (pathway to gang) needed for life to emerge

Nonsense-mediated mRNA reduction and pre-mRNA processing in drosophila

From bacteria to mammalian cells, the presence of a nonsense mutation causes a reduction in the level of the mRNA of the corresponding gene. The reduction is not, contrary to initial expectations, due to a passive mechanism by which non translated mRNAs are degraded; rather it is a active process in which active translation, cis-acting sequences and specific trans-acting factors are required. It is generally accepted that this phenomenon is the consequence of an evolutionary conserved mechanism that evolved to protect cells from the potentially deleterious effect of truncated proteins - this is often referred to as the mRNA surveillance system or nonsense mediated mRNA decay (NMD). This phenomenon has been extensively studied in budding yeast and in mammalian systems and to a lesser extent in C. elegans. In yeast the recognition of the nonsense codon appears to occur during cytoplasmic translation and premature translation termination is thought to activate a specific protein complex - called the surveillance complex - which in tum triggers an accelerated decay of the aberrant mRNA. However, contrary to the expectation that the recognition of the nonsense codon should occur during cytoplasmic translation, several studies in mammalian cells indicate that NMD may take place in the nucleus by a mechanism that is independent of cytoplasmic translation. For example, several reports indicate that this reduction occurs while the mRNA is still associated with the nucleus, and that the stability of the cytoplasmic mRNA is unchanged relative to a wild-type allele. The common view in the field is that these apparently discordant results between NMD in yeast and in mammalian cells will eventually be accommodated in a single model in which translation in the cytoplasm plays a prominent role. For example, a commonly given explanation is that the recognition of the nonsense codon takes place during nuclear export, and it has been implied that the apparent effects on nuclear RNA are in fact triggered by the premature abortion of translation at the cytoplasmic side of the nuclear envelope. However not all the data from mammalian systems can be so easily explained by the above model. For example, several reports indicate that nonsense mutations affect the splicing of the corresponding pre-mRNA, which makes it difficult to imagine how premature translation in the cytoplasm could effect such an early event in mRNA biogenesis

Nonsense Mediated Decay Associated Pioneer Round of Translation as Source for Peptides for Presentation by MHC Class I

Nonsense mediated decay (NMD) plays a critical role in the mRNA quality control mechanism protecting cells from aberrant mRNAs containing premature termination codons (PTC). Recognition of these PTCs requires a pioneer round of translation prior to the bulk translation of mRNAs for cellular protein synthesis, therefore producing a pool of immediate early peptides. Effectiveness of the immune surveillance system is insured by the timely and complete MHC I mediated presentation of a pool of precisely cleaved peptides representing all cellular proteins. So far sources of antigenic peptides are known to include regular degraded proteins, defective ribosomal products (DRiPs) and cryptic peptides starting with a non-AUG start codon. Here the contribution of peptides originating from the pioneer translation to the pool of peptides presented by MHC I molecules is described. Depletion of the essential NMD factor hUpf1 as well as the pioneer translation initiation complex factors CBP80 and CBP20 reveal impaired MHC I antigen presentation. In sharp contrast, targeted interference with bulk translation without effecting pioneer translation permits antigen presentation. Taken together these findings put products of the pioneer round of translation into an important position as novel source for antigenic peptides and reveal a new relationship between NMD and immune surveillance

Characterization of posttranscriptional regulation elements – From protein degradation to functional RNA structures

Posttranscriptional gene regulation in eukaryotic cells is one of the most important mechanism in complex life to either control protein synthesis or diversity at any given moment in the life of a cell. Consequently, deregulation of the many processes involved in posttranscriptional regulation can lead to severe diseases, such as Alzheimer’s disease or certain types of cancer. RNA-binding proteins (RBPs) can also facilitate alternative splicing (AS), which increases protein diversity and fine tunes the protein amount and function in the case of different stress conditions. A deregulation of AS events can lead to severe diseases, such as Parkinson’s disease or certain cancer variants. I characterized a newly described mechanism, where AS events are coupled to rapid protein decay. We termed this event AS-CPD, alternative splicing coupled to constitutive protein decay. The protein decay signal (degron) found in mammalian cells is also functional in Saccharomyces (S.) cerevisiae and Escherichia (E.) coli cells. It is dependent on hydrophobic amino acids in the C-terminus and has the capability to rapidly and efficiently reduce the amount of the tagged protein to an undetectable amount in mere minutes. This mechanism might be due to a conserved stress response in the tested organism. The degron described in this thesis could be a potential tool for new kinetic analysis. Major players in posttranscriptional regulation are trans-acting RBPs, such as Roquin or AUF1. Roquin is a key regulator in immune homeostasis and recognises stem-loop (SL) structures in 3’-UTRs (untranslated regions) to destabilise certain messenger RNAs (mRNAs). In this study, we found, that Roquin can recognise not only classical constitutive decay elements (CDEs), but also AU-rich decay element (ARE)-like CDEs. The binding of these elements may also be subject to competition between different RBPs, as it is in the case of the UCP3 3’-UTR CDEs. Here, Roquin and AUF1 compete for the binding to CDE1. RNA structure often implies a certain function in the organism. The pandemic causing virus SARS-CoV-2 is an RNA virus with a 30 kb long heavily structured genome. The many SLs in the 5’- and 3’-UTRs fulfil different essential functions, which are partially unknown. Here, we provide a detailed analysis of the structure of the genome and impose a screen of chemical compounds with the ability to bind RNA. We found a potent binder D01 with the ability to bind the pseudoknot in between the two open reading frames (ORFs) 1a and 1b, which encode different and essential parts of the viral proteome. These compounds might be a precursor for future potent drugs, able to target RNA viruses with structured genomes

Cancer microenvironment and endoplasmic reticulum stress response

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma

Functional analysis of the JIL-1 histone H3 kinase in Drosophila

Epigenetic regulations play a crucial role in control of gene expression and development. Histone modification is one of the epigenetic mechanisms that contribute to regulation of chromatin structure and gene expression. In Drosophila melanogaster, JIL-1, the predominant interphase histone H3S10 kinase, localizes specifically to euchromatic interband regions of polytene chromosomes, and is upregulated two-fold on the male X chromosome. Genetic interaction assays with JIL-1 hypomorphic and null allelic combinations demonstrated that JIL-1 can counterbalance the gene-silencing effect of the three major heterochromatin components Su(var)3-9, Su(var)3-7, and HP1a on position-effect variegation. Our data suggested that the epigenetic H3S10ph mark functions to counteract heterochromatic spreading and gene silencing in Drosophila . JIL-1 can be divided into four main domains including a NH2-terminal domain (NTD), the first kinase domain (KDI), the second kinase domain (KDII), and a COOH-terminal domain (CTD). Transgenic analysis demonstrated that the CTD of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes, but that both COOH- and NH2-terminal sequences were necessary for upregulation on the male X chromosome. Another construct ∆CTD that lacks the CTD domain but has histone H3S10 kinase activity can be localized to chromatin by the NTD and was able to rescue autosomes as well as partially rescue male X polytene chromosome morphology. Furthermore, to study the role of histone H3S10 phosphorylation in transcription we examined the distribution of JIL-1 and histone H3S10 phosphorylation under both heat shock and non-heat shock conditions. There was no redistribution or upregulation of JIL-1 or histone H3S10 phosphorylation found at transcriptionally active puffs after heat shock treatments. Also in JIL-1 null mutant backgrounds, heat shock-induced puffs were strongly labeled by the antibody to the elongating form of RNA polymerase II (Pol IIoser2), indicating that Pol IIoser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. These results suggested a model where transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin rather than direct effects on Pol II activation. To further study the interplay between JIL-1 and transcription regulation, a genome-wide analysis of JIL-1 kinase binding sites by ChIP-seq was conducted and combined with an analysis of whole genome transcription level changes by RNA-seq in the absence of JIL-1. We found that most of the identified JIL-1 binding peaks locate around 200 bp upstream of transcription start sites and that in the absence of H3S10 phosphorylation by JIL-1 a number of normally actively expressed genes were repressed, whereas some inactive genes were activated. Moreover, in the absence of JIL-1 the gene expression level changes of two JIL-1 target genes showed alterations in H3K9 dimethylation levels. Taken together, all these observations suggested a model that H3S10 phosphorylation mainly facilitates gene expression of active genes by maintaining an open chromatin structure at promoter regions by counteracting heterochromatization

Molecular basis of the human ApoAII exon 3 splicing

Among human genes, CFTR intron Vlll/exon 9 and apolipoprotein All intron Il/exon 3 boundaries share the characteristic feature given by the presence of a peculiar tract of alternating pyrimidines and purines close to the 3’ splice site. In the case of CFTR gene, the pyrimidine rich tract is composed by a stretch of Ts in a row and a stretch of alternating pyrimidines and purines (microsatellite TG dinucleotide repeats) and both tracts are polymorphic for their length. On the other hand, in the case of ApoAII gene the pyrimidine rich tract is made exclusively of alternating pyrimidines and purines (microsatellite TG dinucleotide repeats) and it is also polymorphic for its length in both genes. This apparent sequence similarity, concerning the TG tract, is contrasted by the different splicing pattern exhibited by the two genes. In fact, CFTR exon 9 undergoes alternative splicing to a variable extent, depending on the variations in length of the pyrimidine rich tract, whereas ApoAII exon 3 is constitutively included in mRNA. Previous studies of our group have shown in the CFTR intron Vlll/exon 9 context, the stretch of pyrimidines alternated with purines (within the UG tract) alone is not equivalent to a functional continuous polypyrimidine tract, contrarily to what has been observed for the apolipoprotein All gene. Moreover, the comparison of splice sites strength of human ApoAII exon 3 and human CFTR exon 9 has outlined an apparent contradiction between the splicing behavior of the two exons and the strength of the splice sites. In fact, both the 3’ and the 5’ splice sites of CFTR exon 9 display a good match with the consensus whereas the match of the ApoAII exon 3 splice sites is not good. Altogether these observations prompted us to investigate the mechanisms underlying the constitutive splicing of ApoAII exon 3 and, in particular, to characterize the cis-acting elements and the trans-acting factors involved in ApoAII exon 3 definition to assure its constitutive splicing. In order to study in vivo the splicing mechanism of ApoAII exon 3, we set up an eukaryotic expression system by cloning the whole ApoAII gene, from its promoter to the poly-A signal. Then, the effects of point mutations, deletions or substitutions on splicing of exon 3 were analyzed by RT-PCR after transient transfection in Hep3B cell line. Deletion or replacement of the UG repeats at the 3’ splice site of intron 2 resulted in a significant increase in exon 3 skipping, indicating the importance of this alternated arrangement of U and G as a functional polypyrmidine tract or at least as an important sequence able to lead the exon 3 definition. Furthermore, UV-crosslinking assays showed that the (UG)I6 repeats of ApoAII intron 2 are recognized by TDP-43, a protein that binds specifically the UG tract within the context of the 3' end of CFTR intron VIII and that affects negatively CFTR exon 9 splicing. Next, we characterized the exonic cis-acting elements able to affect the splicing efficiency of ApoAII exon 3. Transient transfections of different constructs of the ApoAII gene system carrying deletions or point mutations showed that the region spanning from nucleotide 87 to 113 of human ApoAII exon 3 is important for its close to the 5’ splice site. In order to identify trans-acting factor/s able to bind the 9nt-ESE within ApoAII exon 3, both Electro Mobility Shift Assay (EMSA) and UV-crosslinking coupled to immunoprécipitation assays were carried out. EMSA showed a broad band of shifted material with the ESEwt RNA, whereas no significant shift was seen with mutated ESE RNA. This suggested that one or more proteins interact specifically with the wild type ApoAII exon 3 across the 9nt-sequence. Then, UV-crosslinking followed by immunoprécipitation with monoclonal antibodies anti-SR proteins ASF/SF2 and anti-SC-35 showed that ESEwt but not mutated ESE RNA was able to immunoprecipitate a band whose molecular weight corresponds to that of ASF/SF2 and, even if to a lower extent, also a band whose molecular weight corresponds to that of SC35. Thus, these results provided an evidence that at least two SR proteins are able to interact with the sequence across the ESE sequence. Subsequently, the relevance of ESE position within an internal intron (and therefore with other flanking cis-acting elements) was also tested. Both in vitro and in vivo experiments showed that the ApoAII exon 3 ESE works only if it is localized within an internal exon and not if it is placed within the first or the last exon. These results also suggested the presence of other splicing regulatory elements within the flanking intronic regions of ApoAII exon 3. Thus, to explore intron 2 and 3 for the presence of cis-acting elements able to affect the splicing of exon 3, a series of deletions within both introns were carried out. Thus, we found at least one regulatory element placed within intron 3 that regulates positively exon 3 inclusion. In conclusion, the constitutive splicing of ApoAII exon 3 seems to be the result of the balance between positive and negative action of the regulatory elements found in the exon 3 and its flanking introns. Future studies will be aimed at identifying the factors interacting with the intronic regulatory elements and at defining a possible model to explain the mechanism of ApoAII exon 3 constitutive splicing by integrating the network of interactions among the identified cis-acting elements and trans-acting factors

HIV rev-isited

The human immunodeficiency virus type 1 (HIV-1) proteome is expressed from alternatively spliced and unspliced genomic RNAs. However, HIV-1 RNAs that are not fully spliced are perceived by the host machinery as defective and are retained in the nucleus. During late infection, HIV-1 bypasses this regulatory mechanism by expression of the Rev protein from a fully spliced mRNA. Once imported into the nucleus, Rev mediates the export of unprocessed HIV-1 RNAs to the cytoplasm, leading to the production of the viral progeny. While regarded as a canonical RNA export factor, Rev has also been linked to HIV-1 RNA translation, stabilization, splicing and packaging. However, Rev's functions beyond RNA export have remained poorly understood. Here, we revisit this paradigmatic protein, reviewing recent data investigating its structure and function. We conclude by asking: what remains unknown about this enigmatic viral protein