Farberov, S. | Search bioRxiv

bioRxiv2024

Systematic analysis of the target recognition and repression by the Pumilio proteins

RNA binding proteins orchestrate the post-transcriptional fate of RNA molecules, but the principles of their action remain poorly understood. Pumilio (PUM) proteins bind 3UTRs of mRNAs and lead to mRNA decay. To comprehensively map the determinants of recognition of sequences by PUM proteins in cells and to study the binding outcomes, we developed a massively parallel RNA assay that profiled thousands of PUM binding sites in cells undergoing various perturbations or RNA immunoprecipitation. By studying fragments from the NORAD long noncoding RNA, we find two features that antagonize repression by PUM proteins - G/C rich sequences, particularly those upstream of the PUM recognition element, and binding of FAM120A, which limits the repression elicited by PUM binding sites. We also find that arrays of PUM sites separated by 8-12 bases offer particularly strong repression and use them to develop a particularly sensitive reporter for PUM repression. In contrast, PUM sites separated by shorter linkers, such as some of those found in NORAD, exhibit strong activity interdependence, likely mediated by competition between PUM binding and formation of strong secondary structures. Overall, our findings expand our understanding of the determinants of PUM protein activity in human cells. HighlightsO_LIA massively parallel assay reports on the binding and activity of Pumilio proteins in human cells C_LIO_LIG/C rich sequence context inhibits repression by Pumilio proteins C_LIO_LIFAM120a binds sequences with Pumilio sites and antagonizes repression by Pumilio proteins C_LIO_LIArrays of Pumilio binding sites are most effective at distances of 8-12 nt. C_LIO_LIStrong inter-dependency in the tandem Pumilio binding sites in NORAD. C_LI

molecular biology↗

bioRxiv2021

Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

molecular biology↗