Project description:RNA guanine quadruplexes (rG4s) are non-canonical nucleic acid structures essential for various cellular functions and disease pathways. Characterizing rG4-interacting proteins (rG4IPs) is crucial for deciphering biological roles of rG4s. Building upon the RNA-protein interaction detection (RaPID) method, we introduced G4-RaPID – a tailored strategy for robustly profiling rG4IPs in living cells. Using G4-RaPID with three distinct rG4 sequences in live cells, we identified 105 candidate rG4IPs with diverse biological functions. We also validated the direct binding of recombinant hnRNPA0, CHD4, and IGF2BP1 proteins with rG4 structures in vitro. Additionally, our CLIP-Seq data revealed an enrichment of hnRNPA0 at rG4 loci. Moreover, luciferase reporter assay results showed that hnRNPA0 interacts with the rG4 in the 5UTR of NRAS mRNA to negatively regulate its translation. Together, we developed an rG4-RaPID method for characterizing rG4-protein interactions in living cells, and documented the functions of hnRNPA0-rG4 interaction in modulating the translation of NRAS mRNA.
Project description:The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.
Project description:RNA-seq on HepG2 cells treated with an shRNA knockdown against HNRNPA0. (HNRNPA0-BGHLV22) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf
Project description:Integrase CLIP-seq experiments were conducted on wild-type and eccentric HIV-1 virions generated in the presence of allosteric integrase inhibitors and IN K264/266A and R269/K273A mutations Integrase CLIP-seq experiments were conducted by immunoprecipitation of integrase-RNA complexes from fully formed mature and eccentric virus particles. Libraries of RNA molecules bound by integrase were generated and sequenced by Illumina Hi-Seq2000 and 2500 platforms.
Project description:UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are the most frequently used methods to study protein-RNA interactions in the intact cells and tissues, but their relative advantages or inherent biases have not been evaluated. To benchmark CLIP and iCLIP method, we performed iCLIP with Nova protein, which is the most extensively studied protein by CLIP. Further, we assessed UV-C-induced cross-linking preferences, by exploiting the UV-independent formation of covalent RNA cross-links of the mutant RNA methylase NSUN2.
