Helical defects in MicroRNA influence protein binding by TAR RNA binding protein

Roderico Acevedo, Nichole Orench-Rivera, Kaycee A. Quarles, Scott A. Showalter

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background: MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly. Methodology/Principal Findings: We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding. Conclusion/Significance: We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream.

Original languageEnglish (US)
Article numbere0116749
JournalPloS one
Volume10
Issue number1
DOIs
StatePublished - Jan 21 2015

Fingerprint

RNA-binding proteins
Double-Stranded RNA
protein binding
double-stranded RNA
MicroRNAs
microRNA
Protein Binding
Defects
RNA
Proteins
RNA-Induced Silencing Complex
Peptides
Regulator Genes
Circular Dichroism
polypeptides
Gene expression
trans-activation responsive RNA-binding protein
Assays
circular dichroism spectroscopy
multiprotein complexes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Acevedo, Roderico ; Orench-Rivera, Nichole ; Quarles, Kaycee A. ; Showalter, Scott A. / Helical defects in MicroRNA influence protein binding by TAR RNA binding protein. In: PloS one. 2015 ; Vol. 10, No. 1.
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Helical defects in MicroRNA influence protein binding by TAR RNA binding protein. / Acevedo, Roderico; Orench-Rivera, Nichole; Quarles, Kaycee A.; Showalter, Scott A.

In: PloS one, Vol. 10, No. 1, e0116749, 21.01.2015.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Helical defects in MicroRNA influence protein binding by TAR RNA binding protein

AU - Acevedo, Roderico

AU - Orench-Rivera, Nichole

AU - Quarles, Kaycee A.

AU - Showalter, Scott A.

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N2 - Background: MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly. Methodology/Principal Findings: We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding. Conclusion/Significance: We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream.

AB - Background: MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly. Methodology/Principal Findings: We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding. Conclusion/Significance: We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream.

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