Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Abstract

RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

Directed evolution of split APEX peroxidase

“…Given the purportedly broad array of novel RNAs and RNA-mediated processes that have eluded mechanistic dissection 82 and the growing number of diseases now thought to be mediated by aberrant RNAprotein interactions 83,84 , there is a great need for identifying the interaction partners of specific RNAs. Conventional approaches address this problem by capturing and enriching a target RNA along with its bound interaction partners, either using affinity-tagged antisense oligonucleotides that isolate endogenous transcripts [85][86][87][88] , or by affinity tagging the transcript of interest itself 89,90 . However, these approaches are often confounded by the abundance of the target transcript, the unpredictable performance of RNA-based affinity tags 91 , and nonspecific RNA-protein interactions formed during lysis and enrichments 92 .…”

Protein sequestration as a normal function of long noncoding RNAs and a pathogenic mechanism of RNAs containing nucleotide repeat expansions

“…al. 2013) and can bind a large number of RNA binding proteins with proposed roles in dysregulation of the normal biological function of these sequestered proteins (Table 2) (reviewed in Jazurek et. al.…”

Reduction of Huntington’s Disease RNA Foci by CAG Repeat-Targeting Reagents

“…Mutant mRNA is partially or temporarily retained in the nucleus within splicing speckles, as shown in multiple types of cellular models of polyQ diseases (De Mezer et al, 2011; Urbanek et al, 2016). Increased retention of mutant RNA in the nucleus is associated with compromised functions of proteins that bind to mutant RNA (Jazurek et al, 2016); examples of such malfunctions are alternative splicing abnormalities (Mykowska et al, 2011; Sathasivam et al, 2013; Cabrera and Lucas, 2016) and other gene expression alterations (Sharma and Taliyan, 2015). For that reason, RNA foci are increasingly considered undesired toxic structures, rather than a protective cellular self-defense mechanism.…”