Quantitative Proteomics to Identify Nuclear RNA-Binding Proteins of Malat1.

Marian Scherer,Marion Scheibe,Falk Butter,Michal Levin

doi:10.3390/ijms21031166

Marian Scherer, Marion Scheibe + Show 2 more

Open Access

https://doi.org/10.3390/ijms21031166

Copy DOI

Abstract

The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function together with RNA-binding proteins (RBPs), the composition of their RBP complex can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5′-region and one in the 3′-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.

Highlights

Mass spectrometry-based proteomics allows identifying thousands of proteins in a single experiment and has been instrumental for screens to identify unknown interaction partners in affinity purification experiments
Alternative approaches provided catalogues of RNA-binding proteins (RBPs) purified from in vivo cross-linked RNA combined with poly-A capture [3,4] and have been complemented by techniques like capture hybridization analysis of RNA targets and mass spectrometry (CHART-MS), comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), and RNA antisense purification and mass spectrometry (RAP-MS) [5,6,7,8] that affinity capture a single target RNA from the in vivo cellular environment using hybridization probes
We identified 35 proteins to be able to interact with nuclear Malat1

Summary

Introduction

Mass spectrometry-based proteomics allows identifying thousands of proteins in a single experiment and has been instrumental for screens to identify unknown interaction partners in affinity purification experiments. Alternative approaches provided catalogues of RNA-binding proteins (RBPs) purified from in vivo cross-linked RNA combined with poly-A capture [3,4] and have been complemented by techniques like capture hybridization analysis of RNA targets and mass spectrometry (CHART-MS), comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), and RNA antisense purification and mass spectrometry (RAP-MS) [5,6,7,8] that affinity capture a single target RNA from the in vivo cellular environment using hybridization probes These and other proteomics strategies have been applied to non-coding RNAs [9]. The MALAT1 lncRNA precursor transcript in humans has a length of 8779 nt (refseq: NR_002819) and is processed into a full-length lncRNA transcript of ca. 8 kb that localizes to nuclear speckles [14] and a 61 nt mascRNA (MALAT1-associated small cytoplasmic RNA) that functions in the cytoplasm [15]

Methods

Results

Conclusion