Valerie de Crecy-Lagard

Professor 

Description of Research

Global vision

The study and implementation of “accuracy” is the general theme unifying the different components of my research program. Accuracy of the translation process with the study of the role of tRNA modifications in cellular physiology. Accuracy of metabolism with the discovery of novel mechanisms of metabolite repair. Finally, accuracy of annotations with my constant preoccupation on improving functional annotations of genomes.

Another common thread that links the different research projects in my laboratory is the use of comparative genomic and experimental methods to link gene and function (summarized in the figure below) that has allowed us to discover the function of over fifty protein families covering several hundred thousand individual genes in the last fifteen years. This in silicodriven approach has also placed me at the interface between the computer and experimental sciences and between the classical genetic and biochemical communities and the bioinformatic and annotation ones.

Specific research projects

Role of complex RNA modification in integration of translation and metabolism. I realized over fifteen years ago that the field of RNA modification was a goldmine for finding “missing genes” by comparative genomic approaches. The modifications had been discovered in the 1970’s but many of the corresponding enzymes/genes never identified. I focused in my laboratory on filling this knowledge gap with an emphasis on deciphering the pathways for the most complex modifications, those that require more than one enzymatic step such as Q, t6A, yW and G+.  We were successful in this endeavor and the biochemical pathways for these modifications are now nearly fully understood (see1,2for reviews).  In the process, we annotated ~30 families previously of unknown function and set the stage to understand the function of complex tRNA modifications in cellular physiology. Indeed, the absence of the universal t6A modification leads to complex and varied phenotypes 3,4.  The modification is essential in Bacteria and Archaea but not in yeast opening the door to the development of antibacterial targets, but the actual cause of this essentiality is not clear. We are currently exploring the detailed molecular mechanisms of how the absence of t6A leads to a wide range of cellular defects ranging from cell division deficiencies to enhanced protein glycation in different bacterial models and in yeast. 

 For the Q modification, we encountered the opposite problem. Even if this modification is widespread in the bacterial kingdom, mutants lacking this modification do not have obvious phenotypes. Using comparative genomic analyses, a link between Q and metal homeostasis was made that led to the discovery of a novel phenotype: E. coli mutants that lack Q are more sensitive to excess cadmium and more resistant to nickel than wild-type cells. The molecular bases for these phenotypes are not yet understood and under exploration but they do confirm the links predicted in silico. We have conducted transcriptomics studies of the WT and Q deficient strain and are planning proteomics analyses to decipher the causes of the nickel resistance phenotype.

 Finally, comparative genomic analyses led to the discovery of Q-like modifications in genomic DNA of bacteria and phages 5. These previously undetected complex modifications are part of novel anti-restriction or restriction modification mechanisms that are we now characterizing.

 

Discovery of novel tRNA repair mechanisms. In Eukaryotes tRNAs that lack specific modifications can be degraded by specific RNAses6. Such a tRNA quality control and/or regulatory mechanisms have yet to be identified in prokaryotes. Preliminary comparative genomic and experimental evidence suggests that novel tRNA surveillance pathways do exists in prokaryotes and that tRNA modifications participate in these pathways. We are combining analytical tools developed by Peter Dedon with biochemical and genetic tools to explore potential new roles of modification in tRNA quality control and novel regulation processes in prokaryotes.  In my laboratory, we have conducted a synthetic lethal screen in Escherichia coliby crossing all viable tRNA modifications genes deletions. Around 10 % of the 400 constructed crosses gave rise to negative genetic interactions and the molecular basis for these phenotypes are currently under study.  

 

Discovery of novel metabolite damage repair or preemption mechanisms.  One great surprise from sequencing genomes of thousands of different organisms is that these genomes code for hundreds of enzymes whose functions are completely unknown and whose existence was never suspected. Our bioinformatic analyses, taken with literature data, show that many of these mystery enzymes probably function to fix mistakes made by metabolic pathways. These mistakes cause damage to small molecules (aka metabolites) in cells, and give rise to abnormal molecules (damaged metabolites) that are at best useless and at worst toxic. The mistakes that cause metabolite damage come in two flavors: those made by enzymes acting on the wrong molecules, and those that come from chemical instability, which causes some molecules to rearrange or break down on their own.  The enzymes that deal with metabolite damage work by fixing the damage product (damage repair) or by avoiding it by turning dangerous metabolites into harmless ones (damage pre-emption) (see 7,8for reviews).   Many of these metabolite repair mechanisms are conserved across kingdoms from E. colito human and deficiencies in these mechanisms can lead to diseases. A specific example is the YggS family. This ubiquitous PLP binding family is of unknown function and a combination of bioinformatics, genetic and analytic experiments suggest that it has a role in PLP  homeostasis  9,10that could prevent damage from occurring by formation of Knoevenageladducts  with intermediates of proline or lysine metabolism. This would fall in the category of damage prevention described above.

 

Quality annotations; the next “big data” challenge of the post-genomic era.  It is quite obvious to all users of genome data that the quality of functional annotations of genes is poor 11. The consensus from different estimates is that over half of the functional annotations are inexistent (such as protein of unknown function), vague (such as ATPase) or outright wrong. In addition, the quality of annotation is not getting better but worse as the number of genomes explodes and error propagates12.  In general, both the funding and experimental communities have not taken this problem seriously preferring to fund data generation rather that data curation. The situation could be much improved by using a combination of comparative genomic analysis with expert knowledge and there has been a recent realization that if nothing is done, the genomic data will become less and less useful. I have done different analyses revealing the poor status of functional annotations and have proposed some solutions11,13,14. This led to my inclusion in different working groups at Argonne National Laboratories, NCBI and Uniprot that are trying to improve the status-quo, bring more biologists in the annotation process and create funding avenues for improving annotations

References

  1. El Yacoubi, B., Bailly, M. & de Crécy-Lagard, V. Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Ann Rev Genet46,69–95 (2012).
  2. Phillips, G. & de Crécy-Lagard, V. tRNA modification in Archaea. Cur. Opin. Microbiol.(2011).
  3. Thiaville, P. C. et al.Essentiality of threonylcarbamoyladenosine (t(6) A), a universal tRNA modification, in bacteria. Mol. Microbiol.98,1199–1221 (2015).
  4. Thiaville, P. C. & de Crecy-Lagard, V. The emerging role of complex modifications of tRNA in signaling pathways. Microb. cell2,1–4 (2015).
  5. Thiaville, J. J. et al.Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc Natl Acad Sci USA113,E1452–E1459 (2016).
  6. Parker, R. RNA Degradation in Saccharomyces cerevisae. Genetics191,671–702 (2012).
  7. Linster, C. L., Van Schaftingen, E. & Hanson, A. D. Metabolite damage and its repair or pre-emption. Nat. Chem. Biol.9,72–80 (2013).
  8. Hanson, A. D., Henry, C. S., Fiehn, O. & de Crécy-Lagard, V. Metabolite Damage and Metabolite Damage Control in Plants. Annu. Rev. Plant Biol.67,131–52 (2016).
  9. Prunetti, L. et al.Evidence That COG0325 proteins are involved in PLP Homeostasis. Microbiology162,694–706 (2016).
  10. Darin, N. et al.Mutations in PROSC disrupt cellular pyridoxal phosphate homeostasis and cause vitamin-B6-dependent epilepsy. Am. J. Hum. Genet.99,1325–1337 (2016).
  11. de Crécy-Lagard, V. Quality annotations, a key frontier in the microbial sciences.Microbe Mag.doi:doi:10.1128/microbe.11.303.1
  12. Schnoes, A. M., Brown, S. D., Dodevski, I. & Babbitt, P. C. Annotation error in public databases: misannotation of molecular function in enzyme superfamilies. PLoS Comput Biol5,e1000605 (2009).
  13. de Crécy-Lagard, V. Variations in metabolic pathways create challenges for automated metabolic reconstructions: Examples from the tetrahydrofolate synthesis pathway. Comput. Struct. Biotechnol. J.10,41–50 (2014).
  14. Zallot, R., Harrison, K. J., Kolaczkowski, B. & de Crécy-Lagard, V. Functional Annotations of Paralogs: A Blessing and a Curse. Life (Basel, Switzerland)6,39 (2016).
Teaching Interests
  • BSC 4434c - Introduction to Bioinformatics
  • BSC 4913 and 4914 - Research in Bioinformatics
  • MCB 6318 - Comparative Microbial Genomics
  • MCB 6940 - Microbiology Career Seminar
  • Bioinformatics Minor Coordinator

Valerie de Crecy-Lagard