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RNA. 2015 Nov;21(11):1921-30. doi: 10.1261/rna.051227.115. Epub 2015 Sep 9.

Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4.

Author information

1
Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
2
Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
3
Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
4
Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Abstract

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.

KEYWORDS:

CRISPR; Csy4; gene regulation; mRNA stability; translation

PMID:
26354771
PMCID:
PMC4604432
DOI:
10.1261/rna.051227.115
[Indexed for MEDLINE]
Free PMC Article

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