Pseudouridine and N⁶-methyladenosine modifications weaken PUF protein/RNA interactions

Abstract

RNA modifications are ubiquitous in biology, with over 100 distinct modifications. While the vast majority were identified and characterized on abundant noncoding RNA such as tRNA and rRNA, the advent of sensitive sequencing-based approaches has led to the discovery of extensive and regulated modification of eukaryotic messenger RNAs as well. The two most abundant mRNA modifications-pseudouridine (Ψ) and -methyladenosine (mA)-affect diverse cellular processes including mRNA splicing, localization, translation, and decay and modulate RNA structure. Here, we test the hypothesis that RNA modifications directly affect interactions between RNA-binding proteins and target RNA. We show that Ψ and mA weaken the binding of the human single-stranded RNA binding protein Pumilio 2 (hPUM2) to its consensus motif, with individual modifications having effects up to approximately threefold and multiple modifications giving larger effects. While there are likely to be some cases where RNA modifications essentially fully ablate protein binding, here we see modest responses that may be more common. Such modest effects could nevertheless profoundly alter the complex landscape of RNA:protein interactions, and the quantitative rather than qualitative nature of these effects underscores the need for quantitative, systems-level accounting of RNA:protein interactions to understand post-transcriptional regulation.

mRNA Traffic Control Reviewed: N6‐Methyladenosine (m6A) Takes the Driver's Seat

“…The accessibility of hPUM2 – a PUF family ssRNA binding protein that binds to an m 6 A‐modified oligomer (CCUGUA m UA m UA m U) is reduced by 30‐fold compared to the affinity of unmodified RNA. While certain RNA modifications abrogate RNA binding affinity of proteins, m 6 A modification demonstrates a modest inhibition of hPUM2 binding which warrants further studies . Further studies are required to shed light on novel anti‐readers and their co‐ordination with m 6 A.…”

RNAs, Phase Separation, and Membrane‐Less Organelles: Are Post‐Transcriptional Modifications Modulating Organelle Dynamics?

“…For instance, YTH domain‐containing proteins are specifically attracted to m 6 A in RNA . m 6 A can act as a “molecular switch” by causing the disruption of particular structural motifs, thereby regulating the accessibility for RNA‐binding proteins . For instance, U‐rich sequences that interact with major constituents of SGs (i.e., hnRNPs) are frequently masked by poly‐A stretches, which can be substrates for m 6 A methylation.…”

Pre‐mRNA modifications and their role in nuclear processing

“…In another example, pseudouridine indirectly influenced PRP5 binding to the U2 snRNA by stabilizing a structure, the branchpoint-interacting stem loop [67], a mechanism that could also happen in mRNA. Additionally, binding of the cytoplasmic RNA-binding protein Pumilio 2 to its UGUAR binding motif was modestly decreased when the second uridine was pseudouridine [119]. UGYAR is relatively abundant among endogenously pseudouridy- lated mRNAs due to pseudouridylation by PUS7 in the same context [3].…”