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Directed protein evolution of an RNA editor for single molecule RNA-binding protein co-occupancy (II)

ABSTRACT: RNA base editors have become powerful tools for both therapeutic and research purposes. However, currently available RNA editors such as ADAR or APOBEC family proteins have limitations due to RNA structure and sequence dependence. Here, we designed a protein engineering platform for putative RNA editor screening and directed enzyme evolution. We engineered an RNA A-to-I base editor (rABE) that has high activity, low local sequence or structure bias, and low background. Utilizing rABE, we present REMORA (RNA Encoded Molecular Recording in Adenosines), a new strategy to measure RNA-binding events on single RNA molecules in cells. In REMORA, adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by mutations by sequencing. By combining our improved A-to-I RNA deaminase with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, our approach enables simultaneous recording of the locations two RNA binding proteins on single mRNA molecules. Orthogonal RNA molecular recording of two Pumilio family proteins, PUM1 and PUM2, reveals that PUM1 competes with PUM2 for some but not all Pumilio binding sites in cells, despite having the same in vitro binding preferences. Our work thus measures competition between RNA-binding proteins for RNA sites in cells, and our genetically encodable RNA deaminase enables single-molecule identification of RNA-protein interactions with cell type specificity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE241615 | GEO | 2023/08/24

REPOSITORIES: GEO

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Directed protein evolution of an RNA editor for single molecule RNA-binding protein co-occupancy (I)

Project description:RNA base editors have become powerful tools for both therapeutic and research purposes. However, currently available RNA editors such as ADAR or APOBEC family proteins have limitations due to RNA structure and sequence dependence. Here, we designed a protein engineering platform for putative RNA editor screening and directed enzyme evolution. We engineered an RNA A-to-I base editor (rABE) that has high activity, low local sequence or structure bias, and low background. Utilizing rABE, we present REMORA (RNA Encoded Molecular Recording in Adenosines), a new strategy to measure RNA-binding events on single RNA molecules in cells. In REMORA, adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by mutations by sequencing. By combining our improved A-to-I RNA deaminase with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, our approach enables simultaneous recording of the locations two RNA binding proteins on single mRNA molecules. Orthogonal RNA molecular recording of two Pumilio family proteins, PUM1 and PUM2, reveals that PUM1 competes with PUM2 for some but not all Pumilio binding sites in cells, despite having the same in vitro binding preferences. Our work thus measures competition between RNA-binding proteins for RNA sites in cells, and our genetically encodable RNA deaminase enables single-molecule identification of RNA-protein interactions with cell type specificity.

2022-10-28 | GSE216334 | GEO

Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

Project description:Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 function to negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the DCP2 mRNA decapping enzyme has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent pathway.

2021-09-09 | GSE159510 | GEO

The interactome of flavin adenine dinucleotide (FAD) with RNA-binding proteins

Project description:We explored the interactome of flavin adenine dinucleotide (FAD) and the matrix of UV-immobilised FAD bound more than 3,000 proteins from SW-620 lysate. GO enrichment analysis showed that about 600 RNA-binding proteins were bound to FAD beads and, surprisingly, more than 40 proteins showed clear dose-dependent binding competition with nanomolar affinities indicating that their direct or indirect interaction with free FAD is both specific and strong. Among the few known consensus sequences for RNA-binding proteins, we selected the PUF motif 5'-UGUANAUA-3' to investigate whether FAD is binding the Pumilio homologs PUM1 and PUM2 in their RNA-binding pocket, as both these proteins showed dose-response behaviour in the FAD versus FAD-beads. Immobilisation of an oligomer containing the consensus sequence on NHS-activated Sepharose beads yielded PUM-beads that did enrich the Pumilio homologs. Oligomer vs FAD-beads and FAD vs PUM-beads together with the FAD vs FAD-beads dose-response competition indicated that the binding of FAD does not seem to influence the binding of the RNA. It is therefore most likely that FAD binding is allosteric for PUMs.

2022-10-06 | PXD036953 | Pride

Gene expression profiles in NORAD knockout and PUMILIO overexpressing cells

Project description:Purpose: PUMILIO proteins are known to repress target genes by specifically binding to PUMILIO response elements (PREs) in target mRNAs. NORAD is a noncoding RNA that negatively regulates PUMILIO activity. The goal of this study was to determine the gene expression changes that result from knockout of NORAD or overexpression of PUMILIO and to test whether NORAD knockout causes PUMILIO hyperactivity. Methods: RNA-seq libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat Sample Preparation kit (Illumina) and sequenced using the 100 bp paired-end protocol on an Illumina HiSeq 2000. For comparing NORAD+/+ and NORAD-/- HCT116 cells, 3 biological replicates per genotype were sequenced. For PUM overexpression experiments, 3 replicates of GFP-expressing HCT116 cells (negative control) and 2 independent PUM1- or PUM2-overexpressing clones (2 replicates each) were sequenced. Results: Gene expression profiles show that PUMILIO target genes are downregulated in both NORAD knockout cells and PUMILIO overexpressing cells. Conclusions: These data indicate that NORAD sequesters PUMILIO, preventing excessive repression of PUMILIO target genes that are important for maintaining genomic stability.

2015-12-03 | GSE75440 | GEO

Identification of RNAs bound to endogenous PUM2 in HCT116 cells

Project description:Purpose: PUMILIO proteins are known to repress target genes by binding to PUMILIO response elements (PREs) in target mRNAs. The goal of this study was to demonstrate binding of endogenous PUM2 to the noncoding RNA NORAD and to identify PUM2 target genes in NORAD wild-type and knockout HCT116 cells. Methods: PAR-CLIP was performed with endogenous PUM2 in HCT116 cells and isogenic NORAD knockout cells. Results: Endogenous PUM2 binds to NORAD in HCT116 cells. In addition, PUM2 target genes were identified in HCT116 cells. Conclusions: PUM2 binds to NORAD through multiple PREs on NORAD. Compared to all other PUM2 target genes in HCT116, NORAD is the preferred binding partner of endogenous PUM2.

2015-12-03 | GSE75439 | GEO

Mammalian Pumilio Proteins Control Cellular Morphology, Migration, and Adhesion

Project description:Pumilio proteins are RNA-binding proteins that control mRNA translation and stability by binding to the 3' UTR of target mRNAs. Mammals have two canonical Pumilio proteins, PUM1 and PUM2, which are known to act in many biological processes, including embryonic development, neurogenesis, cell cycle regulation and genomic stability. Here, we characterized a new role of both PUM1 and PUM2 in regulating cell morphology, migration, and adhesion in T-REx-293 cells, in addition to previously known defects in growth rate. Gene ontology analysis of differentially expressed genes in PUM double knockout (PDKO) cells for both cellular component and biological process showed enrichment in categories related to adhesion and migration. PDKO cells had a collective cell migration rate significantly lower than that of WT cells and displayed changes in actin morphology. In addition, during growth, PDKO cells aggregated into clusters (clumps) due to an inability to escape cell-cell contacts. Addition of extracellular matrix (Matrigel) alleviated the clumping phenotype. Collagen IV (ColIV), a major component of Matrigel, was shown to be the driving force in allowing PDKO cells to monolayer appropriately, however, ColIV protein levels remained unperturbed in PDKO cells. This study characterizes a novel cellular phenotype associated with cellular morphology, migration, and adhesion which can aid in developing better models for PUM function in both developmental processes and disease.

2023-01-01 | GSE207836 | GEO

A global identification of PUM1 and PUM2 mRNA targets and their protein cofactors in human seminoma TCam-2 cells

Project description:The functions of human PUM1 and PUM2 are considered to be redundant given that both PUF1 and PUF2 recognize the same PBE motif UGUANAUA. However pools of mRNAs published so far for PUM1 and PUM2 do not overlap. Therefore we sought to investigate the issue of redundancy in human cells. The both PUM proteins are less conserved in the region that is outside the PUM domain. We sought that interactors could be different. We identified mRNA pools binding separately PUM1 and PUM2 by RIP-Seq approach, normalized them using the whole TCam-2 cells transcriptome and aligned them with mRNA pools activated or repressed as tested by siRNA PUM1 and PUM2 knockdown.

2018-11-28 | GSE123016 | GEO

Principles of mRNA control by human Pumilio proteins elucidated from global transcriptome stability, in vitro affinity profiling, and integrative data analysis

Project description:The human members of the PUF family of proteins, PUM1 and PUM2, are RNA-binding proteins that post-transcriptionally regulate gene expression through binding to a PUM recognition element (PRE) in the 3′ UTR of target mRNAs, promoting RNA decay. Recent RNA-seq experiments in PUM1/2 knockdown conditions have identified hundreds of known and new human PUM targets through measurement of changes in steady state RNA levels. However, steady-state RNA levels do not allow for measurement of changes in RNA stability between conditions and do not allow for the differentiation between the contributions of changes in transcription rates and changes in RNA decay. Here, we identify hundreds of human PUM1/2 targets that have changes in RNA stability following PUM1/2 knockdown. We separate the contributions of changes in transcription rate and RNA stability and find that human PUM proteins almost exclusively modulate RNA abundance through changing RNA stability and not transcription. In addition, we find that the sequence preferences for all possible 8mers are largely similar between PUM1 and PUM2, suggesting that PUM1 and PUM2 recognize similar targets. We identify an ideal PRE “rulebook” by determining key contextual features around PREs, including local AU content, location of a PRE within a 3′ UTR, clustering of PREs, and number of miRNA sites near a PRE, that help differentiate functional PREs from non-functional ones as measured by our decay dataset. Consistent with previously identified functional roles of mammalian PUMs, we find that human PUM1 and PUM2 modulate the decay of genes related to signaling cascades and neuronal function. Finally, we train machine learning models to predict functional regulation of RNA targets by the human PUM proteins and find that contextual features around PREs contribute meaningful information to our models.

2020-08-05 | GSE145237 | GEO

A conserved abundant cytoplasmic long noncoding RNA modulates repression of mRNAs by Pumilio proteins in human cells

Project description:Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionary conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD - an abundant and highly conserved cytoplasmic lncRNA. Most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two Pumilio homologs in mammals. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA binding proteins, an activity which positions them at key junctions of cellular signaling pathways.

2016-07-11 | GSE79804 | GEO

Post-Transcriptional Regulation of Mouse Neurogenesis by Pumilio Proteins [RIP-Seq]

Project description:we employ crosslinking immunoprecipitation (iCLIP) to reveal Pum1 and Pum2 binding mRNAs

2017-08-15 | GSE95195 | GEO