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Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4.

Publication ,  Journal Article
Borchardt, EK; Vandoros, LA; Huang, M; Lackey, PE; Marzluff, WF; Asokan, A
Published in: RNA
November 2015

The bacterial CRISPR endoribonuclease Csy4 has recently been described as a potential RNA processing tool. Csy4 recognizes substrate RNA through a specific 28-nt hairpin sequence and cleaves at the 3' end of the stem. To further explore applicability in mammalian cells, we introduced this hairpin at various locations in mRNAs derived from reporter transgenes and systematically evaluated the effects of Csy4-mediated processing on transgene expression. Placing the hairpin in the 5' UTR or immediately after the start codon resulted in efficient degradation of target mRNA by Csy4 and knockdown of transgene expression by 20- to 40-fold. When the hairpin was incorporated in the 3' UTR prior to the poly(A) signal, the mRNA was cleaved, but only a modest decrease in transgene expression (∼2.5-fold) was observed. In the absence of a poly(A) tail, Csy4 rescued the target mRNA substrate from degradation, resulting in protein expression, which suggests that the cleaved mRNA was successfully translated. In contrast, neither catalytically inactive (H29A) nor binding-deficient (R115A/R119A) Csy4 mutants were able to exert any of the effects described above. Generation of a similar 3' end by RNase P-mediated cleavage was unable to rescue transgene expression independent of Csy4. These results support the idea that the selective generation of the Csy4/hairpin complex resulting from cleavage of target mRNA might serve as a functional poly(A)/poly(A) binding protein (PABP) surrogate, stabilizing the mRNA and supporting translation. Although the exact mechanism(s) remain to be determined, our studies expand the potential utility of CRISPR nucleases as tools for controlling mRNA stability and translation.

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Published In

RNA

DOI

EISSN

1469-9001

Publication Date

November 2015

Volume

21

Issue

11

Start / End Page

1921 / 1930

Location

United States

Related Subject Headings

  • RNA, Messenger
  • RNA Stability
  • Protein Biosynthesis
  • Protein Binding
  • Poly A
  • Inverted Repeat Sequences
  • Humans
  • HEK293 Cells
  • Endoribonucleases
  • Developmental Biology
 

Citation

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MLA
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Borchardt, E. K., Vandoros, L. A., Huang, M., Lackey, P. E., Marzluff, W. F., & Asokan, A. (2015). Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4. RNA, 21(11), 1921–1930. https://doi.org/10.1261/rna.051227.115
Borchardt, Erin K., Leonidas A. Vandoros, Michael Huang, Patrick E. Lackey, William F. Marzluff, and Aravind Asokan. “Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4.RNA 21, no. 11 (November 2015): 1921–30. https://doi.org/10.1261/rna.051227.115.
Borchardt EK, Vandoros LA, Huang M, Lackey PE, Marzluff WF, Asokan A. Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4. RNA. 2015 Nov;21(11):1921–30.
Borchardt, Erin K., et al. “Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4.RNA, vol. 21, no. 11, Nov. 2015, pp. 1921–30. Pubmed, doi:10.1261/rna.051227.115.
Borchardt EK, Vandoros LA, Huang M, Lackey PE, Marzluff WF, Asokan A. Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4. RNA. 2015 Nov;21(11):1921–1930.

Published In

RNA

DOI

EISSN

1469-9001

Publication Date

November 2015

Volume

21

Issue

11

Start / End Page

1921 / 1930

Location

United States

Related Subject Headings

  • RNA, Messenger
  • RNA Stability
  • Protein Biosynthesis
  • Protein Binding
  • Poly A
  • Inverted Repeat Sequences
  • Humans
  • HEK293 Cells
  • Endoribonucleases
  • Developmental Biology