Identification of the genetic mechanism that associates L3MBTL3 to multiple sclerosis.

Multiple sclerosis (MS) is a complex and demyelinating disease of the central nervous system. One of the challenges of the post-genome-wide association studies (GWAS) era is to understand the molecular basis of statistical associations to reveal gene networks and potential therapeutic targets. The L3MBTL3 locus has been associated with MS risk by GWAS. To identify the causal variant of the locus, we performed fine mapping in a cohort of 3440 MS patients and 1688 healthy controls. The variant that best explained the association was rs6569648 (P = 4.13E-10, odds ratio = 0.71, 95% confidence interval (CI) = 0.64-0.79), which tagged rs7740107, located in intron 7 of L3MBTL3. The rs7740107 (A/T) variant has been reported to be the best expression and splice quantitative trait locus (eQTL and sQTL) of the region in up to 35 human genotype-tissue expression (GTEx) tissues. By sequencing RNA from blood of 17 MS patients and quantification by digital qPCR, we determined that this eQTL/sQTL originated from the expression of a novel short transcript starting in intron 7 near rs7740107. The short transcript was translated into three proteins starting at different translation initiation codons. These N-terminal truncated proteins lacked the region where L3MBTL3 interacts with the transcriptional regulator Recombination Signal Binding Protein for Immunoglobulin Kappa J Region which, in turn, regulates the Notch signalling pathway. Our data and other functional studies suggest that the genetic mechanism underlying the MS association of rs7740107 affects not only the expression of L3MBTL3 isoforms, but might also involve the Notch signalling pathway.

Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes.

Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.

The miR-302-367 cluster as a potential stemness regulator in ESCs.

Increasing experimental evidence suggests an important role of miRNAs in embryonic stem cell (ESC) biology. The miR-302-367 cluster is exclusively expressed at high levels in ESCs but not in either somatic stem cells or adult/embryonic differentiated cells. The human miR-302-367 gene structure has been recently described and its promoter has been identified, characterized and functionally validated in human stem cells. The miR-302-367 promoter activity depends on the ontogeny and hierarchical cellular stage. The miR-302-367 promoter is transcriptionally regulated by the ESC-specific transcription factors Oct3/4, Sox2 and Nanog and, its activity restricted to the ESC compartment. Functionally, this cluster regulates cell cycle in ESCs promoting self-renewal and pluripotency, therefore representing a master regulator in the maintenance of hESC stemness. We envision this data may open up new avenues to investigate the transcriptional regulators upstream miR-302-367 cluster and to dissect the complex interplay by which this miR-302-367 cluster integrates in the molecular network conferring pluripotency to ESCs. In this perspective, we summarize recent progress in the genomic and functional characterization of the miR-302-367 cluster and discuss its potential as a stemness determinant.

Inhibition of HIV-1 replication by RNA targeted against the LTR region.

OBJECTIVE: The use of small RNA molecules able to effect gene inactivation has emerged as a powerful method of gene therapy. These small inhibitory RNAs are widely used for silencing malignant cellular and viral genes. We have assayed a series of inhibitory RNAs named catalytic antisense RNAs, consisting of a catalytic domain, hairpin or hammerhead ribozyme, and an antisense domain. The aim of the present study was to evaluate the effect of these inhibitory RNAs on HIV-1 replication. METHODS: A series of expression vectors has been constructed for the intracellular synthesis of inhibitory RNAs, differing in the promoter that drives their synthesis. These inhibitory RNAs were designed to act at two possible cleavage sites in the long terminal repeat (LTR) region and the TAR domain was chosen as a target for the antisense domain. We have evaluated the effects of different inhibitory RNAs in HIV replication via changes in p24 antigen levels. Mobility shift assays have been used to check the binding capacity of inhibitory RNAs. RESULTS: Catalytic antisense RNA designed to target the LTR region of HIV-1 inhibited viral replication in an eukaryotic cell environment by more than 90%. The conventional hairpin and hammerhead ribozymes, however, failed to inhibit viral replication. CONCLUSIONS: The data provide preliminary evidence of a new class of inhibitory RNAs that can be used to block HIV replication. The results clearly show the importance of the ex vivo antisense effect in the inhibition achieved. A good correlation was found between the in vitro binding efficiency of the inhibitor RNA to the HIV-1 LTR and the inhibition of viral replication.

HIV-1 TAR as anchoring site for optimized catalytic RNAs.

Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence. We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain. A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop. The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme. Deletion of the TAR domain in the substrate abolishes the positive effect. These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs. A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs. Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs. Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.