Project description:To detect the modifed bases in SINEUP RNA, we compared chemically modified in vitro transcribed (IVT) SINEUP-GFP RNA and in-cell transcribed (ICT) SINEUP RNA from SINEUP-GFP and sense EGFP co-transfected HEK293T/17 cells. Comparative study of Nanopore direct RNA sequencing data from non-modified and modified IVT samples against the data from ICT SINEUP RNA sample revealed modified k-mers positions in SINEUP RNA in the cell.
Project description:To analyze SINEUP RNA secondary structure in living cells, multiple SINEUP RNA and target EGFP mRNA plasmids were transfected in HEK293T/17 cells and icSHAPE libraries were prepared.
Project description:Loss-of-function mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are strongly associated with Autism Spectrum Disorders (ASD). Indeed, the reduction of CHD8 causes transcriptional, epigenetic and cellular phenotypic changes, correlated to disease that can be monitored in assessing new therapeutic approaches. SINEUPs are a functional class of natural and synthetic antisense long non-coding RNAs able to stimulate the translation of antisense target mRNA, with no effect on transcription. Here we employed synthetic SINEUP-CHD8 targeting the first and third AUG of the CHD8 coding sequence to efficiently stimulate endogenous CHD8 protein production. SINEUP-CHD8 were effective in cells with reduced levels of the target protein and in patients’-derived fibroblasts with CHD8 mutations. Functionally, SINEUP-CHD8 were able to revert molecular phenotypes associated with CHD8-suppression, i.e. genome-wide transcriptional dysregulation, and the reduction of H3K36me3 levels. Strikingly, in chd8-morpholino-treated and ENU mutant zebrafish embryos, SINEUP-chd8 injection confirmed the ability of SINEUP RNA to rescue the chd8-suppression-induced macrocephaly phenotype and neuronal hyperproliferation. Thus, SINEUP-CHD8 molecule(s) represent a proof-of-concept towards the development of a novel RNA-based therapy for neurodevelopmental syndromes with implications for, and beyond ASD, and relevant to genetic disorders caused by protein haploinsufficiency.
Project description:The covalent modification of RNA molecules is a pervasive feature of all classes of RNAs and has fundamental roles in the regulation of several cellular processes. Mapping the location of RNA modifications transcriptome-wide is key to unveiling their role and dynamic behaviour, but technical limitations have often hampered these efforts. Nanopore direct RNA sequencing is a third-generation sequencing technology that allows the sequencing of native RNA molecules, thus providing a direct way to detect modifications at single-molecule resolution. Despite recent advances, the analysis of nanopore sequencing data for RNA modification detection is still a complex task that presents many challenges. Many works have addressed this task using different approaches, resulting in a large number of tools with different features and performances. Here we review the diverse approaches proposed so far and outline the principles underlying currently available algorithms.
Project description:Long-read direct RNA sequencing developed by Oxford Nanopore Technologies (ONT) is quickly gaining popularity for transcriptome studies, while fast turnaround time and low cost make it an attractive instrument for clinical applications. There is a growing interest to utilize transcriptome data to unravel activated biological processes responsible for disease progression and response to therapies. This trend is of particular interest for precision medicine which aims at single-patient analysis. Here we evaluated whether gene abundances measured by MinION direct RNA sequencing are suited to produce robust estimates of pathway activation for single sample scoring methods. We performed multiple RNA-seq analyses for a single sample that originated from the HepG2 cell line, namely five ONT replicates, and three replicates using Illumina NovaSeq. Two pathway scoring methods were employed-ssGSEA and singscore. We estimated the ONT performance in terms of detected protein-coding genes and average pairwise correlation between pathway activation scores using an exhaustive computational scheme for all combinations of replicates. In brief, we found that at least two ONT replicates are required to obtain reproducible pathway scores for both algorithms. We hope that our findings may be of interest to researchers planning their ONT direct RNA-seq experiments.
Project description:We found novel functional long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA, named SINEUPs. To investigate the network of translational regulation, we focused on the sub-cellular distribution of target mRNAs and SINEUP RNAs and SINEUP RNA binding proteins (RBPs). We identified PTBP1 and HNRNPK as essential RBPs. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhancement of the target mRNA translation. To prove the SINEUP RBPs binding regions on SINEUP-GFP transcripts, we performed seCLIP; single-end enhanced crosslinking and immunoprecipitation assay to determine the specific binding sites of PTBP1 and HNRNPK on SINEUP-GFP RNA. These findings will promote a better understanding of the mechanisms on the fate of regulatory RNAs implicated in efficient protein translation.