scholarly journals Effective RNA Regulation by Combination of Multiple Programmable RNA-Binding Proteins

2020 ◽  
Vol 10 (19) ◽  
pp. 6803
Author(s):  
Misaki Sugimoto ◽  
Akiyo Suda ◽  
Shiroh Futaki ◽  
Miki Imanishi
Keyword(s):  
Expression Vector ◽  
Binding Proteins ◽  
Protein Targeting ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Luciferase Reporter ◽  
Cooperative Effects ◽  
Puf Proteins ◽  
Puf Protein ◽  
Target Rna

RNAs play important roles in gene expression through translation and RNA splicing. Regulation of specific RNAs is useful to understand and manipulate specific transcripts. Pumilio and fem-3 mRNA-binding factor (PUF) proteins, programmable RNA-binding proteins, are promising tools for regulating specific RNAs by fusing them with various functional domains. The key question is: How can PUF-based molecular tools efficiently regulate RNA functions? Here, we show that the combination of multiple PUF proteins, compared to using a single PUF protein, targeting independent RNA sequences at the 3′ untranslated region (UTR) of a target transcript caused cooperative effects to regulate the function of the target RNA by luciferase reporter assays. It is worth noting that a higher efficacy was achieved with smaller amounts of each PUF expression vector introduced into the cells compared to using a single PUF protein. This strategy not only efficiently regulates target RNA functions but would also be effective in reducing off-target effects due to the low doses of each expression vector.

scholarly journals Combinatorial recognition of clustered RNA elements by a multidomain RNA-binding protein, IMP3

2018 ◽  
Author(s):  
Tim Schneider ◽  
Lee-Hsueh Hung ◽  
Masood Aziz ◽  
Anna Wilmen ◽  
Stephanie Thaum ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Binding Domains ◽  
Systematic Strategy ◽  
Rna Elements ◽  
Target Rna

AbstractHow multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

A Cooperative Mechanism of Target RNA Selection via Germ Cell-Specific RNA-Binding Proteins, NANOS2 and DND1

2021 ◽  
Author(s):  
Takamasa Hirano ◽  
Danelle Wright ◽  
Atsushi Suzuki ◽  
Makoto Kiso ◽  
Yumiko Saga
Keyword(s):  
Germ Cell ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Rna

scholarly journals New approaches to target RNA binding proteins

2021 ◽  
Vol 62 ◽  
pp. 13-23
Author(s):  
Ashley R. Julio ◽  
Keriann M. Backus
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
New Approaches ◽  
Target Rna

scholarly journals The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

2020 ◽  
Author(s):  
Ryota Kurimoto ◽  
Tomoki Chiba ◽  
Yoshiaki Ito ◽  
Takahide Matsushima ◽  
Yuki Yano ◽  
...  
Keyword(s):  
Binding Proteins ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Luciferase Reporter ◽  
Transcriptional Gene Silencing ◽  
Stem Loop ◽  
Pseudouridine Synthase ◽  
Wide Range ◽  
And Function

AbstractLet-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter, and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, HITS-CLIP (High throughput sequencing crosslinking immunoprecipitation) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR-8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation and function.

scholarly journals Ribonomic Analysis of Human Pum1 Reveals cis-trans Conservation across Species despite Evolution of Diverse mRNA Target Sets

2008 ◽  
Vol 28 (12) ◽  
pp. 4093-4103 ◽  
Author(s):  
Adam R. Morris ◽  
Neelanjan Mukherjee ◽  
Jack D. Keene
Keyword(s):  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Identification ◽  
Consensus Sequence ◽  
Puf Proteins ◽  
Mrna Targets ◽  
Puf Protein ◽  
Genes Encoding

ABSTRACT PUF family proteins are among the best-characterized regulatory RNA-binding proteins in nonmammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1-associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the posttranscriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3′ untranslated region sequences of Pum1-associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and relocalization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1-associated mRNAs to mRNA targets of PUF proteins from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates how a well-conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes.

scholarly journals Roles of Plant Glycine-Rich RNA-Binding Proteins in Development and Stress Responses

2021 ◽  
Vol 22 (11) ◽  
pp. 5849
Author(s):  
Liqun Ma ◽  
Ke Cheng ◽  
Jinyan Li ◽  
Zhiqi Deng ◽  
Chunjiao Zhang ◽  
...  
Keyword(s):  
Stress Responses ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Recognition Motif ◽  
Functional Roles ◽  
Rich Domain ◽  
C Terminus ◽  
Cold Shock Domain ◽  
Target Rna

In recent years, much progress has been made in elucidating the functional roles of plant glycine-rich RNA-binding proteins (GR-RBPs) during development and stress responses. Canonical GR-RBPs contain an RNA recognition motif (RRM) or a cold-shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus, which have been associated with several different RNA processes, such as alternative splicing, mRNA export and RNA editing. However, many aspects of GR-RBP function, the targeting of their RNAs, interacting proteins and the consequences of the RNA target process are not well understood. Here, we discuss recent findings in the field, newly defined roles for GR-RBPs and the actions of GR-RBPs on target RNA metabolism.

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