Assistant Professor of

Biochemistry and Molecular Biology

Ph.D. (1991) Harvard University

RNA Helicases, mRNA Degradation Mechanisms

 Tel: (305) 243-7229, Fax: (305) 243-3955

cjain@med.miami.edu

We are interested in understanding RNA metabolism in a model organism, the bacterium Escherichia coli.  RNA metabolism is defined as a series of events that commence with the incorporation of mononucleotides into an RNA chain and conclude with the degradation of RNA into its constituent nucleotides.  We work with E. coli because we believe that a detailed understanding of such fundamental life processes is most easily attained in a relatively simple and well-characterized organism.  At present, the current emphasis in the lab is on two topics: RNA helicases and RNA degradation mechanisms in E. coli. 

RNA helicases

The ability of cellular factors to interact with RNA is critically dependent upon the structure of the latter.  RNA structure can be altered by enzymes known as RNA helicases, which unravel RNA duplexes, and are present in most organisms.  Among the most prominent class of RNA helicases are those that belong to the DEAD-box family.  Despite the progress in defining their biochemical properties, the cellular functions of DEAD-box helicases are still poorly understood in most organisms.   We are using a combination of biochemical and genetic approaches to understand the function of most of the five E. coli DEAD-box RNA helicases.  Specific projects that are ongoing in the lab are listed below. 

 Defining helicase function We have identified genetic suppressors of the growth defects caused by the absence of specific helicases in the cell.  A preliminary analysis of the suppressors indicates that these are involved in multiple aspects of RNA function, including translation, RNA modification and RNA processing.  We are currently investigating the mechanism by which suppression occurs, which we hope will provide insights into the molecular functions of the helicases.

 Interactions with cellular transcripts  We have identified several E. coli transcripts that are up-regulated or down-regulated in strains that lack specific helicases.  We believe that many of these RNAs are substrates for the helicases, which change the conformation of these RNAs and render them differentially sensitive to the action of ribonucleases (RNases).  The mechanisms by which helicases affect the abundance of these RNAs is currently being investigated. 

 Interactions with Poly(A) polymerase (PAP)  In E. coli, many RNAs are modified at their 3’ end by the addition of poly(A) residues, a process catalyzed by PAP.   Such RNAs are recognized more efficiently by RNases, and hence, are degraded faster.  We have identified a novel functional interaction between PAP and the E. coli DEAD-box RNA helicases, which stimulates PAP activity in vitro.  The consequences of this interaction for PAP function and RNA degradation in vivo are being addressed.

RNA degradation mechanisms

 In many organisms, mRNAs are rapidly degraded.  Rapid mRNAs turnover is due to the presence of RNases, which have been conserved through evolution and are present in all organisms. This topic is of special significance, not only because it comprises a major aspect of RNA metabolism, but also because the instability of mRNAs has major consequences for protein expression.  Some questions of interest include the following:

How is mRNA turnover regulated?  It is well known that transcription of many genes is regulated in response to changes in the cellular environment.  However, there is very little known about the regulation of RNA degradation, which is a complementary process.  We would like to conduct a genome-wide survey of RNA half-lives, investigate how this parameter changes in response to different environments, and identify any regulatory factors responsible for these changes.

Is there channeling between the RNases in the Degradosome?  Most mRNAs are first cleaved by RNase E, an endonuclease.  RNase E also occurs in a complex called the “Degradosome”, along with the exonuclease Polynucleotide phosphorylase (PNPase) and other factors.  It is hypothesized that Degradosome-mediated mRNA degradation occurs through a channeled set of events: first, cleavage by RNase E, followed by transfer of the cleaved fragments to PNPase for further digestion.  We would like to investigate whether the different steps in mRNA breakdown are coordinated in the proposed manner.

Recent Publications

 ·         Jain, C (2006). Overexpression and purification of tagged Escherichia coli proteins using a chromosomal knock-in strategy. Protein Expr. Purif. 46, 294-98.

·         Briegel, K. J., Baker, A and Jain, C.  (2006) Identification and analysis of Escherichia coli ribonuclease E dominant-negative mutants.  Genetics 172, 7-15.

·         Slagter-Jäger, J. G., Puzis, L., Gutgsell,^, N. S., Belfort,^, M., and Jain, C.  (2007)^. Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors. RNA 13, 597-605.

·         Yang, J., Jain, C. and Schesser, K. (2008). RNase E regulates the Yersinia type 3 secretion system. J Bacteriol. 190, 3774-8

·         Jain C (2008).  The E. coli RhlE RNA helicase regulates the function of related RNA helicases during ribosome assembly. RNA 14, 381-9.

A postdoctoral position is available. Please inquire by email!