RNA World/Scientific objectives/en
- The CRISPR elements are part of a prokaryotic defence system directed against external attacks by e.g. viruses and may be viewed as a simple immune system of microorganisms.
By employing RNA World to systematically screen organisms for the presence of the various types of this defence machinery, we hope to acquire important information on the global distribution and varieties of this system. There is an enormous repertoire of potential applications to the results of such analyses ranging from the improvement of industrially relevant microbial food production to novel ways of coping with multi-drug resistant pathogenic bacteria.
- 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 Mar 23;315(5819):1709-12.
- Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure. 2009 Jun 10;17(6):904-12.
- Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009 Nov 25;139(5):945-56.
- Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 2010 Feb 2.
- The GEMM RNA motif is a so-called cis-acting riboswitch designed to detect the second messenger cyclic di-GMP. The bottom line is that GEMM is an upstream terminal part of an mRNA that can bind to this small signal molecule. Upon binding, its structure is modulated such that the downstream part of the mRNA which encodes a protein is affected in such a way that, depending on the type of GEMM motif, the corresponding protein production is either activated or inactivated. Hence, GEMM serves as an RNA-based molecular switch to control protein production.
The reason for why we are interested in this RNA module is twofold: (1) its switch properties make it very useful for synthetic biology applications and (2) GEMM controls production of proteins that are essential for the capability of certain pathogens to attack human cells. Examples for such pathogens are the cholera-causing bacterium Vibrio cholerae or Bacillus anthracis which is responsible for anthrax. What RNA World does is a systematical screen of all completely sequenced organisms in order to identify this GEMM motif.
- Sudarsan N, Lee ER, Weinberg Z, Moy RH, Kim JN, Link KH, Breaker RR. Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science. 2008 Jul 18;321(5887):411-3.
- Smith KD, Lipchock SV, Ames TD, Wang J, Breaker RR, Strobel SA. Structural basis of ligand binding by a c-di-GMP riboswitch. Nat Struct Mol Biol. 2009 Dec;16(12):1218-23.
- 6S RNA is a small non-coding RNA of about 200 nucleotides in size which is widespread among bacteria and occasionally even occours in multiple gene copies within a given species. It appears to represent a system to preserve RNA polymerase under conditions of nutrient limitation. Interestingly, due to its special structure, this RNA may serve as a template for bacterial house-keeping RNA polymerase to generate small miRNA-sized RNAs upon nutrient re-supply, thereby demonstrating that RNA polymerases can act as both DNA-dependent RNA polymerases and RNA-dependent RNA polymerases.
In this project we attempt to chart the presence of this regulatory system in all completely sequenced organisms.
- Willkomm DK, Hartmann RK. 6S RNA - an ancient regulator of bacterial RNA polymerase rediscovered. Biol Chem. 2005 Dec;386(12):1273-7.
- Wassarman KM, Saecker RM. Synthesis-mediated release of a small RNA inhibitor of RNA polymerase. Science. 2006 Dec 8;314(5805):1601-3.
- Gildehaus N, Neusser T, Wurm R, Wagner R. Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts. Nucleic Acids Res. 2007;35(6):1885-96.
- Wassarman KM. 6S RNA: a regulator of transcription. Mol Microbiol. 2007 Sep;65(6):1425-31.
- All organisms examined so far to this respect respond to a sudden decrease in ambient temperature by inducing a specialized genetic program termed the cold shock response (CSR). This cellular stress response is designed to cope with a broad variety of temperature-dependent modulations of molecular structures, transport processes, chemical reactivities, and many more.
The goal of this project is to systematically identify non-coding RNAs (ncRNAs) involved in thermoregulation in all organisms whose genomes have been completely sequenced.
- Weber MHW, Marahiel MA. Bacterial cold shock responses. Sci Prog. 2003;86(Pt 1-2):9-75.
Project: Mtb, Myctu, Mycle & Myc
- Mycobacterium tuberculosis is the the causative agent of tuberculosis (TB) which is a world-wide pandemic that is contagious and spreads through the air. Scaringly, more than two billion people, equal to one third of the worldâ??s total population, are infected with TB bacilli. Even worse, multidrug-resistant TB (MDR-TB) is a form of TB that does not respond to the standard treatments using first-line drugs and is present in virtually all countries surveyed by WHO and its partners. Strikingly, a total of 1.77 million people died from TB in 2007, equal to about 4800 deaths a day which makes TB one of the world's major causes of death.
Since it is known that certain non-coding RNAs (ncRNAs) are required to control the ability of many pathogens to infect their hosts, in this project we undertake an exhaustive search to map all ncRNAs known to date in this organism. Moreover, including the leprosy-causing agent Mycobacterium leprae, we extend our bioinformatic analyses to all fully sequenced strains of the genus Mycobacterium and also compare pathogenic versus non-pathogenic strains to possibly identify ncRNA-based differences that might be involved in virulence processes. To validate the biological and medical relevance of our computational investigations, laboratory experiments are performed in cooperation with our research partners in India. It is clear that the potentially possible identification of a ncRNA which is essential for pathogenicity of this organism may represent an excellent novel drug target to battle TB in the future.
- Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998 Jun 11;393(6685):537-44.
- Camus JC, Pryor MJ, Médigue C, Cole ST. Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology. 2002 Oct;148(Pt 10):2967-73.
- Terwilliger TC, Park MS, Waldo GS, Berendzen J, Hung LW, Kim CY, Smith CV, Sacchettini JC, Bellinzoni M, Bossi R, De Rossi E, Mattevi A, Milano A, Riccardi G, Rizzi M, Roberts MM, Coker AR, Fossati G, Mascagni P, Coates AR, Wood SP, Goulding CW, Apostol MI, Anderson DH, Gill HS, Eisenberg DS, Taneja B, Mande S, Pohl E, Lamzin V, Tucker P, Wilmanns M, Colovos C, Meyer-Klaucke W, Munro AW, McLean KJ, Marshall KR, Leys D, Yang JK, Yoon HJ, Lee BI, Lee MG, Kwak JE, Han BW, Lee JY, Baek SH, Suh SW, Komen MM, Arcus VL, Baker EN, Lott JS, Jacobs W Jr, Alber T, Rupp B. The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology. Tuberculosis (Edinb). 2003;83(4):223-49.
- Chhabria M, Jani M, Patel S. New frontiers in the therapy of tuberculosis: fighting with the global menace. Mini Rev Med Chem. 2009 Apr;9(4):401-30.
- Ribozymes are non-coding RNA molecules that, like protein enzymes, catalyze chemical reactions. Examples are the hammerhead ribozyme, the HDV ribozyme, the hairpin ribozyme and many more.
In this project we are screening the kingdom of life for the presence of ribozymes. We also include artificially designed ribozymes in order to find out whether natural analogs exist.
- Tang J, Breaker RR. Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5784-9.
- Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP. RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science. 2001 May 18;292(5520):1319-25.
- Zaher HS, Unrau PJ. Selection of an improved RNA polymerase ribozyme with superior extension and fidelity. RNA. 2007 Jul;13(7):1017-26.
- Lincoln TA, Joyce GF. Self-sustained replication of an RNA enzyme. Science. 2009 Feb 27;323(5918):1229-32. Epub 2009 Jan 8.
Project: tRNA & tRNA-like
- tRNAs are essential components of the cellular protein production machinery but also serve a number of additional functions as e.g. regulating gene expression in conjunction with the T-box element or are utilized as primers for reverse transcription of the HIV genome, a process essential for viral integration into the host genome. Moreover, tRNAs are also required for building the bacterial cell wall and occur in certain lipids making them an extremely versatile molecule family. Interestingly, a number of tRNA-like sequences have been identified in plant virus genomes and appear to be linked to the regulation of viral replication.
In this project, we aim at compiling a complete survey on the occurrence of tRNAs and tRNA-like sequences hoping to identify even more variety in this fundamental molecule class.
- Wegrzyn G, Wegrzyn A. Is tRNA only a translation factor or also a regulator of other processes? J Appl Genet. 2008;49(1):115-22.
- Dreher TW. Role of tRNA-like structures in controlling plant virus replication. Virus Res. 2009 Feb;139(2):217-29. Epub 2008 Jul 30.
- Rich A. The era of RNA awakening: structural biology of RNA in the early years. Q Rev Biophys. 2009 May;42(2):117-37. Epub 2009 Jul 29.
- Pütz J, Giegé R, Florentz C. Diversity and similarity in the tRNA world: overall view and case study on malaria-related tRNAs. FEBS Lett. 2010 Jan 21;584(2):350-8.
- Francklyn CS, Minajigi A. tRNA as an active chemical scaffold for diverse chemical transformations. FEBS Lett. 2010 Jan 21;584(2):366-75.
- The T-box leader is part of the 5' end of a number of mRNAs and represents an RNA element that serves a regulatory function in controlling protein production by interacting with non-charged tRNAs.
In this project, the RNA World supercomputer is utilized to identify T-box elements throughout the kingdom of life.
- Grundy FJ, Rollins SM, Henkin TM. Interaction between the acceptor end of tRNA and the T box stimulates antitermination in the Bacillus subtilis tyrS gene: a new role for the discriminator base. J Bacteriol. 1994 Aug;176(15):4518-26.
- Gerdeman MS, Henkin TM, Hines JV. Solution structure of the Bacillus subtilis T-box antiterminator RNA: seven nucleotide bulge characterized by stacking and flexibility. J Mol Biol. 2003 Feb 7;326(1):189-201.
- Green NJ, Grundy FJ, Henkin TM. The T box mechanism: tRNA as a regulatory molecule. FEBS Lett. 2010 Jan 21;584(2):318-24.
Whenever a new genome sequence is published, its sequence of letters (i.e. the bases of the DNA molecules it contains) is annotated computationally. During this process, all regions are being identified that, based on the standard genetic code and a number of well-known rules, code for proteins. Besides ribosome-related RNAs such as rRNAs and tRNAs and a number of universally occurring non-coding RNAs, the majority of ncRNAs usually escape detection. In this project, we focus on exclusively filling in the gaps resulting from current protein-focused genome annotation procedures by systematically identifying ncRNAs and by adding our results to those generated by the standard methods.