scholarly journals Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

2020 ◽  
Author(s):  
Michael B. Wolfe ◽  
Trista L. Schagat ◽  
Michelle T. Paulsen ◽  
Brian Magnuson ◽  
Mats Ljungman ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Rna Stability ◽  
Decay Rates ◽  
Metabolic Labeling ◽  
Rna Turnover ◽  
Regulate Gene Expression ◽  
Recognition Element ◽  
Rna Targets ◽  
Functional Regulation

AbstractThe human PUF-family proteins, PUM1 and PUM2, post-transcriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3’ UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a ‘rulebook’ of key contextual features that differentiate functional vs. non-functional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

scholarly journals In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

2014 ◽  
Vol 15 (8) ◽  
pp. 14332-14347 ◽  
Author(s):  
Ryan Williams ◽  
Cassandra Crihfield ◽  
Srikanth Gattu ◽  
Lisa Holland ◽  
Letha Sooter
Keyword(s):  
Molecular Recognition ◽  
In Vitro Selection ◽  
Single Stranded Dna ◽  
Recognition Element ◽  
Molecular Recognition Element ◽  
Selection Of

scholarly journals Opposing Effects of Polyadenylation on the Stability of Edited and Unedited Mitochondrial RNAs in Trypanosoma brucei

2005 ◽  
Vol 25 (5) ◽  
pp. 1634-1644 ◽  
Author(s):  
Chia-Ying Kao ◽  
Laurie K. Read
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Stability ◽  
Protein S ◽  
Rna Degradation ◽  
Nucleotide Composition ◽  
Rna Turnover ◽  
Degradation Reactions ◽  
The Stability ◽  
Opposing Effects

ABSTRACT Mitochondrial RNAs in Trypanosoma brucei undergo posttranscriptional RNA editing and polyadenylation. We previously showed that polyadenylation stimulates turnover of unedited RNAs. Here, we investigated the role of polyadenylation in decay of edited RPS12 RNA. In in vitro turnover assays, nonadenylated fully edited RNA degrades significantly faster than its unedited counterpart. Rapid turnover of nonadenylated RNA is facilitated by editing at just six editing sites. Surprisingly, in direct contrast to unedited RNA, turnover of fully edited RNA is dramatically slowed by addition of a poly(A)20 tail. The same minimal edited sequence that stimulates decay of nonadenylated RNA is sufficient to switch the poly(A) tail from a destabilizing to a stabilizing element. Both nucleotide composition and length of the 3′ extension are important for stabilization of edited RNA. Titration of poly(A) into RNA degradation reactions has no effect on turnover of polyadenylated edited RNA. These results suggest the presence of a protective protein(s) that simultaneously recognizes the poly(A) tail and small edited element and which blocks the action of a 3′-5′ exonuclease. This study provides the first evidence for opposing effects of polyadenylation on RNA stability within a single organelle and suggests a novel and unique regulation of RNA turnover in this system.

scholarly journals Terminal Modification, Sequence, and Length Determine Small RNA Stability in Animals

2020 ◽  
Author(s):  
Ildar Gainetdinov ◽  
Cansu Colpan ◽  
Katharine Cecchini ◽  
Paul Albosta ◽  
Karina Jouravleva ◽  
...  
Keyword(s):  
Small Rna ◽  
Viral Infections ◽  
Rna Stability ◽  
Degradation Pathway ◽  
Decay Rates ◽  
List Type ◽  
Piwi Protein ◽  
Rna Modifications ◽  
Regulate Gene Expression ◽  
Pirna Pathway

ABSTRACTIn animals, piRNAs, siRNAs, and miRNAs silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3′ terminal trimming and 2′-O-methylation. Both trimming and methylation influence piRNA stability. Here, we report that trimming and methylation protect mouse piRNAs from different decay mechanisms. In the absence of 2′-O-methylation, mouse piRNAs with extensive complementarity to long RNAs become unstable. In flies, 2′-O-methylation similarly protects both piRNAs and siRNAs from complementarity-dependent destabilization. Animal miRNAs are unmethylated, and complementarity-dependent destabilization helps explain differences in miRNA decay rates in both mice and flies. In contrast, trimming protects mouse piRNAs from a separate degradation pathway unaffected by target complementarity but sensitive to the 3′ terminal, untrimmed sequence. Because distinct sets of mouse piRNAs are protected by each of these mechanisms, loss of both trimming and 2′-O-methylation causes the piRNA pathway to collapse, demonstrating that these two small RNA modifications collaborate to stabilize piRNAs.Highlights2′-O-methylation protects mouse and fly piRNAs from complementarity-dependent decay2′-O-methylation protects fly siRNAs with extensive complementarity to long RNAsComplementarity to long RNAs predicts the half-life of fly and mouse miRNAsMouse pre-piRNA decay reflects both pre-piRNA sequence and PIWI protein identity

scholarly journals Rapid nuclear deadenylation of mammalian messenger RNA

2021 ◽  
Author(s):  
Jonathan Alles ◽  
Ivano Legnini ◽  
Maddalena Pacelli ◽  
Nikolaus Rajewsky
Keyword(s):  
Messenger Rna ◽  
Rna Stability ◽  
Metabolic Labeling ◽  
Cell Fractionation ◽  
Genome Wide ◽  
Cytoplasmic Tails ◽  
Long Read ◽  
Nuclear Synthesis

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition, and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened within minutes after transcription. Our data show that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs escape rapid nuclear deadenylation. Modelling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggest that nuclear deadenylation is a key mechanism for regulating mRNA stability, abundance, and subcellular localization.

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