Araujo Tavares, R. d. C. | Search bioRxiv

bioRxiv2023

Discovery of a Well-Folded Protein Interaction Hub Within the Human Long Non-Coding RNA NORAD

The long non-coding RNA NORAD functions in maintaining genomic stability in humans via sequestering Pumilio proteins from the cytoplasm, and thereby modulating the gene expression of mRNA targets of Pumilio proteins. Despite its role in fundamental cellular pathways including chromosome segregation and DNA damage response, there have been limited structural and biophysical descriptions of NORAD. Here, using an integrative approach combining chemical probing coupled to high throughput sequencing, and RNA-pull downs coupled with mass spectrometry, we discovered a well-folded and structured protein interaction hub within the functional core of NORAD. Our in vitro biochemical reconstitutions using purified recombinant proteins and a NORAD repeat unit region within this hub reveal the assembly of a higher-order multimeric RNA-protein complex.

molecular biology↗

bioRxiv2023

MRT-ModSeq - Rapid detection of RNA modifications with MarathonRT

Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT- ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and modification type. As a proof of concept, we use the MRT fingerprints of well-studied rRNAs to implement a general workflow for detecting RNA modifications. MRT-ModSeq rapidly detects positions of diverse modifications across a RNA transcript, enabling assignment of m1acp3Y, m1A, m3U, m7G and 2-OMe locations through mutation-rate filtering and machine learning. m1A sites in sparsely modified targets, such as MALAT1 and PRUNE1 could also be detected. MRT-ModSeq can be trained on natural and synthetic transcripts to expedite detection of diverse RNA modification subtypes across targets of interest.

biochemistry↗