CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs
Tóm tắt
CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA), which is processed by Cas proteins into small RNA molecules (crRNAs) that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs. We here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA. The simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two orders of magnitude) increase of crRNAs upon cas overexpression. A crucial ingredient of this large increase is fast non-specific degradation by an unspecified nuclease, which suggests that a yet unidentified nuclease(s) is a major control element of CRISPR response. Transcriptional regulation may be another important control mechanism, as it can either increase the amount of generated pre-crRNA, or alter the level of cas gene activity. This article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.
Tài liệu tham khảo
Mojica FJ, Diez-Villasenor C, Soria E, Juez G: Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000, 36: 244-246. 10.1046/j.1365-2958.2000.01838.x.
Jansen R, Embden JD, Gaastra W, Schouls LM: Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002, 43: 1565-1575. 10.1046/j.1365-2958.2002.02839.x.
Horvath P, Barrangou R: CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010, 327: 167-170. 10.1126/science.1179555. (New York, NY)
Jansen R, van Embden JD, Gaastra W, Schouls LM: Identification of a novel family of sequence repeats among prokaryotes. Omics: a journal of integrative biology. 2002, 6: 23-33. 10.1089/15362310252780816.
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD: Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005, 151: 2551-2561. 10.1099/mic.0.28048-0. (Reading, England)
Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E: Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005, 60: 174-182. 10.1007/s00239-004-0046-3.
Pourcel C, Salvignol G, Vergnaud G: CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005, 151: 653-663. 10.1099/mic.0.27437-0. Reading, England
Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV: A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology direct. 2006, 1: 7-10.1186/1745-6150-1-7.
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P: CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007, 315: 1709-1712. 10.1126/science.1138140. New York, NY)
Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R: Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol. 2010, 75: 1495-1512. 10.1111/j.1365-2958.2010.07073.x.
Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, Severinov K: Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol Microbiol. 2010, 77: 1367-1379. 10.1111/j.1365-2958.2010.07265.x.
Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J: Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008, 321: 960-964. 10.1126/science.1159689. New York, NY
Al-Attar S, Westra ER, van der Oost J, Brouns SJ: Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol Chem. 2011, 392: 277-289.
Diez-Villasenor C, Almendros C, Garcia-Martinez J, Mojica FJ: Diversity of CRISPR loci in Escherichia coli. Microbiology. 2010, 156: 1351-1361. 10.1099/mic.0.036046-0. Reading, England
Poranen MM, Ravantti JJ, Grahn AM, Gupta R, Auvinen P, Bamford DH: Global changes in cellular gene expression during bacteriophage PRD1 infection. J Virol. 2006, 80: 8081-8088. 10.1128/JVI.00065-06.
Westra ER, Pul U, Heidrich N, Jore MM, Lundgren M, Stratmann T, Wurm R, Raine A, Mescher M, Van Heereveld L, et al: H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol Microbiol. 2010, 77: 1380-1393. 10.1111/j.1365-2958.2010.07315.x.
Sneppen K: GZ: Physics in Molecular Biology. 2005, Cambridge: Cambridge University Press
Gillespie DT: Stochastic simulation of chemical kinetics. Annu Rev Phys Chem. 2007, 58: 35-55. 10.1146/annurev.physchem.58.032806.104637.
Kruger DH, Schroeder C: Bacteriophage T3 and bacteriophage T7 virus-host cell interactions. Microbiol Rev. 1981, 45: 9-51.
Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K: Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A. 2011, 108: 10098-10103. 10.1073/pnas.1104144108.
Semenova E, Nagornykh M, Pyatnitskiy M, Artamonova II, Severinov K: Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae. FEMS Microbiol Lett. 2009, 296: 110-116. 10.1111/j.1574-6968.2009.01626.x.