Biology subjects

Arnan, C.

Publications and source records attributed to Arnan, C..

3 recordsLinked to original sources

bioRxiv2021

MapToCleave: high-throughput profiling of microRNA biogenesis in living cells

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors, however, they have been conducted in vitro. Here we introduce MapToCleave, a new method to simultaneously profile processing of thousands of distinct RNA structures in living cells. Our new in cell method captures essentially all the biogenesis features that have been discovered through near two decades of in vitro studies - providing support for both approaches. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from twenty animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from ten animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryo-EM studies. In summary, we apply a new screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

molecular biology↗

bioRxiv2021

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify both protein coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that the usage of a second gRNA targeting the same gene synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next take advantage of our paired-guide (pgRNA) system to design a library to simultaneously target 874 pc-genes and 166 lncRNAs which are known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four for deeper characterization. Two of these, FURIN and NFE2, code for proteins related to cell differentiation and macrophage function; the other two, LINC02432 and MIR3945HG, are lncRNAs associated with cancerous and infectious diseases, respectively. The CRISPR-Cas9 coupled to pgRNAs system is, therefore, a suitable tool to target simultaneously pc-genes and lncRNAs for genomic perturbation assays.

genomics↗

bioRxiv2019

bsAS, an antisense long non-coding RNA, controls cell fate through regulation of blistered/DSRF isoform expression

SummaryNatural Antisense Transcripts (NATs) are long non-coding RNAs (lncRNAs) that overlap coding genes in the opposite strand. NATs roles have been related to gene regulation through different mechanisms, including post-transcriptional RNA processing. With the aim to identify NATs with potential regulatory function during fly development, we generated RNA-Seq data in eye-antenna, leg, and wing at third instar larvae. Among the candidate NATs, we found bsAS, antisense to bs/DSRF, a gene involved in wing development and neural processes. Through the analysis of the RNA-Seq data, we found that these two different functions are carried out by the two different protein isoforms encoded in the bs gene. We also found that the usage of these isoforms is regulated by bsAS. This regulation is essential for the correct determination of cell fate during Drosophila development, as bsAS knockouts show highly aberrant phenotypes. bs regulation by bsAS is mediated by the specific physical interaction of the bsAS promoter with the promoters of bs, and it likely involves a mechanism, where expression of bsAS leads to the collision of RNA polymerases acting in opposite directions, preventing the elongation of the longer isoforms of bs, the ones carrying the neural related functions. Evolutionary analysis suggests that the bsAS NAT emerged simultaneously to the long-short isoform structure of bs, preceding the emergence of wings in insects, and maybe related to regulation of neural differentiation.

molecular biology↗