Research Interests: |
| In all
biological systems, RNA molecules--including messenger RNAs (mRNAs), or
noncoding RNAs such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) or
small regulatory RNAs (sRNAs)--serve as intermediates to help regulate
the conversion of the genetic information contained within a cells DNA
into functional proteins. In prokaryotes, such as Escherichia coli,
mRNAs are both synthesized and degraded rapidly, providing the organism
with an excellent mechanism for rapid adaptation to environmental changes.
The half-lives of mRNAs can vary greatly (20 sec-25 min in E. coli),
but generally are not longer than the generation time of the bacterium.
Polycistronic mRNAs are a unique feature in bacteria. Many of these
large transcripts are processed into smaller units as a means of regulating
the expression of specific genes within each operon. For a comprehensive
review of mRNA decay see Kushner (2007). Interestingly, the processing
and decay of noncoding RNAs is carried out by the same enzymes that are
involved in mRNA decay. Thus, the maturation, processing and decay
of tRNAs, rRNAs, and sRNAs are directly related to mRNA decay. Another
fascinating aspect of prokaryotic RNAs is the post-transcriptional addition
of poly(A) tails by poly(A) polymerase I (Mohanty & Kushner, 1999)
and polynucleotide tails by polynucleotide phosphorylase (Mohanty &
Kushner, 2000a). Experiments from our laboratory (O'Hara et al.,
1995, Mohanty & Kushner, 1999) and others have suggested that poly(A)
tails specifically serve as a targeting mechanism for rapid degradation.
My laboratory is currently employing a combination of genetic, molecular biological, genomic, proteomic, bioinformatic, and biochemical approaches to develop a better understanding of the mechanisms involved in the processing, maturation and decay of all types of RNA molecules in E. coli. |
1. Analysis of polyadenylation in Escherichia coli and other prokaryotes |
|
The focus of this project is to delineate the molecular mechanism of polyadenylation
in E. coli. Previous experiments in our laboratory have shown that
poly(A) tails target mRNAs for rapid decay (O'Hara et al., 1995), but also
increase the stability of the mRNAs encoding both RNase E and polynucleotide
phosphorylase (PNPase), two ribonucleases that are involved in degrading
all types of RNA molecules in the cell (Mohanty & Kushner, 1999, Mohanty
& Kushner, 2002). We have also shown that poly(A) polymerase
I is part of a multiprotein complex that also includes PNPase and Hfq,
a small RNA binding protein (Mohanty et al., 2004). In addition,
several lines of experimentation indicate that rho-independent transcription
terminators serve as polyadenylation signals in exponentially growing cells
(Mohanty & Kushner, 2006, Mohanty et al., 2004). A genomic analysis
of the entire E. coli transcriptome has shown that the vast majority
of E. coli ORFs undergo some degree of polyadenylation (Mohanty
&
Kushner, 2006).
However, many questions remain to be answered. For example, why is immature 23S ribosomal RNA a primary substrate for poly(A) polymerase (Mohanty & Kushner, 1999). In addition, the analysis of polyadenylation is complicated by the fact that while poly(A) polymerase accounts for 90% of the poly(A) tails (Mohanty & Kushner, 1999), PNPase also synthesizes polynucleotide tails in wild-type bacteria (Mohanty & Kushner, 2000a, Mohanty et al., 2004). The heteropolymeric tails added by PNPase are generally found near the 5 termini of transcripts and do not appear to perform the same function as poly(A) tails. We are currently trying to determine whether polyadenylation is used primarily to target full-length mRNAs or to help promote the degradation of mRNA fragments generated by endonucleolytic digestion by enzymes such as RNase E, RNase G and RNase III. We are also working to determine if the polyadenylation complex contains additional proteins. Furthermore, we are trying to establish what makes a rho-independent transcription terminator an effective polyadenylation signal. Our current working model of polyadenylation is shown in Fig. 1. |
 |
| Fig. 1. A model for polyadenylation of mRNAs in E. coli. mRNAs with Rho-independent transcription terminators will contain a stem-loop structure at their 3' ends. This structure inhibits the activity of both PNPase and RNase II, the two major 5 -> 3 exonucleases in the cell, because of its very short single-stranded region at the 3 terminus. In the presence of the riboregulator protein Hfq, a complex containing Hfq, PNPase and poly(A) polymerase I (PAP I) binds to the terminus. PAP I then initiates the addition of A residues to form a poly(A) tail. It is not clear whether Hfq remains associated with PAP I after polyadenylation commences. At some point, PAP I is displaced by PNPase and degradation starts in the 3 -> 5 direction, proceeding through the stem-loop structure, releasing the Hfq protein. As PNPase approaches the 5' end of the mRNA, its rate of degradation slows as the localized concentration of inorganic Pi declines. Eventually, the enzyme begins to synthesize a polynucleotide tail on the transcript. A cycling process then ensues until the mRNA is either degraded into a small oligonucleotide (4-7 nt) that is no longer a substrate for PNPase or the enzyme dissociates from the substrate. |
2. Analysis of the multiple mechanisms associated with the processing of tRNA precursors |
| There are 86 tRNA genes in E. coli. They are dispersed throughout the bacterial genome as monocistronic genes or polycistronic loci that contain multiple tRNAs, tRNAs and rRNAs, tRNAs and mRNAs. A variety of data has suggested that RNase E is responsible for the initial separation of all polycistronic transcripts that contained tRNA precursors (Ow & Kushner, 2002, Li & Deutscher, 2002) (Fig. 2). |
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| Fig. 2. General model for tRNA processing in E. coli. In the first step the endoribonuclease RNase E cleaves in the intercistronic regions of polycistronic tRNA precursors to generate pre-tRNAs that have a small number of extra nucleotides at both their 5 and 3 ends. The mature 5 termini are generated by cleavage with the ribozyme, RNase P, while the mature 3 end arises from the action of a series of 3-> 5 exonucleases. The most important of these enzymes are RNase T and RNase PH. |
| While this model explains the initial processing of many tRNA precursors, we have recently shown that there are some polycistronic operons that are processed without any involvement of RNase E. In particular, the valV valW and leuQ leuP leuV operons are separated into pre-tRNAs by RNase P (Mohanty & Kushner, 2007). In this pathway (Fig. 3), the initial cleavages by RNase P generate pre-tRNAs with mature 5 termini after the rho-dependent terminator is removed by a combination of RNase II and PNPase. The processing of the 3 termini is similar to that described in Fig. 2. Subsequently, we have shown that the secG leuU and metT operons also employ RNase P to separate the tRNAs (Mohanty & Kushner, 2008). In the case of the metT operon, which contains seven tRNAs, the processing pathway actually involves both RNase P and RNase E (Mohanty & Kushner, 2008). We are currently studying the maturation of additional tRNAs that use a different mechanism for removing terminator sequences. |
 |
| Fig. 3. Model for the RNase P dependent pathway of tRNA processing in E. coli. In this pathway, the tRNA precursors are initially separated by an RNase P cleavage. The mature 3 termini are generated by the action of RNase T, RNase PH, RNase D, and RNase BN. |
3. Analysis of the role of RNase E in mRNA decay |
| RNase
E (Fig. 4), encoded by rne, was first identified in the late 1970s
based on its involvement in the processing of a 9S rRNA precursor into
a p5S species (Apirion & Lasser, 1978). Subsequently, it was
shown to be involved in mRNA decay (Arraiano et al., 1988) and the maturation
of tRNAs (Ow & Kushner, 2002). Since this enzyme is essential
for cell viability, it was assumed that either the defect in mRNA decay
or 9S rRNA processing was responsible for the loss of cell viability.
However, experiments carried out with a series of RNase E deletion mutations
demonstrated that these assumptions were not correct (Ow et al., 2000).
In fact, experiments employing a series of truncated RNase E proteins led
us to hypothesize that the ability of RNase E to initiate the maturation
of tRNAs was its essential function (Ow & Kushner, 2002). Of
further interest is the fact that RNase E serves as the scaffold for a
multiprotein complex called the "degradosome" (Fig. 4). Since
this complex contains both endo- and exonucleases as well as an RNA helicase
activity, it was assumed that it accounted for the bulk of mRNA decay.
However, analysis of RNase E deletion mutants has demonstrated that assembly
of the degradosome is not required for normal mRNA decay (Ow et al., 2000).
In contrast, degradosome assembly is required for the cell to be able to
detect alterations in the level of polyadenylation (Mohanty & Kushner,
2002).
Additional experiments have shown that the rne gene is controlled by a complex regulatory system that involves three distinct promoters (Ow et al., 2002). With the completion of the crystal structure of the catalytic region of RNase E (Callaghan et al., 2005), it is now possible to examine the interaction of the various domains in the activity of RNase E on various mRNA, tRNA and rRNA substrates. For example, we have recently identified second-site intragenic suppressor mutations that complement the growth defects associated with the rne-1 and rne-3071 alleles at 44oC (Perwez et al., 2008) (Fig. 4). Interestingly, these suppressor mutations restore normal activity on tRNA precursors but mRNA decay remains defective. We are now employing a variety of genetic and biochemical techniques to help understand how RNase E distinguishes among mRNA, rRNA and tRNA substrates. |
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| Figure 4. Model of RNase E showing the catalytic and scaffolding regions of RNase E. The various domains shown in the catalytic region are based on the work of Callaghan et al. (2005). The rne-1 and rne-3071 alleles encode temperature sensitive RNase E proteins. The locations of the intragenic second site suppressors (rne-172, rne-186 and rne-187 are as described in Perwez et al. (2008). |
4. Analysis of the role of RNase G in mRNA decay |
| RNase G is a functional homologue of RNase E that is 34.1% identical over the first 488 amino acids of the RNase E protein (Fig. 5). Many prokaryotes have homologues of at least one of these two proteins. Although deletion of the structural gene for RNase G does not lead to any major phenotypic alterations, it has been shown that the protein is involved in the maturation of 16S rRNA and the decay of a few mRNAs. By constructing a series of isogenic strains containing combinations of the rne-1 and rng::cat alleles, we demonstrated that RNase G can serve as a backup in both the decay of mRNAs and the processing of a 9S rRNA precursor into the 5S rRNA (Ow et al., 2003). However, RNase G was not able to efficiently process tRNA precursors (Ow et al., 2003). Recently, we have recently isolated single-amino acid substitution mutations in the RNase G protein that lead to the ability of RNase G to support cell viability in RNase E deletion mutants. We are currently studying these mutant RNase G proteins at the biochemical level to determine how the catalytic activities have been altered. |
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| Fig. 5. Comparision of the RNase E and RNase G homologs. The diagram of the N-terminus of RNase E is based on the crystallographic analysis of Callaghan et al. (2005). The model for RNase G is based on computer modeling. The location of two single amino acid substitutions in the predicted RNase H domain of RNase G that lead to complementation of RNase E deletion mutants are indicated (Chung et al., 2009). |
5. Analysis E. coli RNA metabolism using high density tiling arrays |
|
Over the past 15 years, we have constructed a variety of
E. coli mutants that are deficient in one or more enzymes thought to be
involved in mRNA decay. In many of these strains we observed reduced
rates of mRNA decay (Arraiano et al., 1988, Babitzke et al., 1993, O'Hara
et al., 1995, Granger et al., 1998, Ow et al., 2003). However, we
have never succeeded in constructing a mutant that was completely defective
in mRNA decay. Thus we have continued to seek additional enzymes
that are involved in the pathways of mRNA decay.
Recently two new endoribonucleases have been identified that play a role in the initiation of mRNA decay in E. coli. The first enzyme is called RNase Z. Originally identified in plants and archaea (Schierling et al., 2002, Schiffer et al., 2002), this enzyme has been shown to be involved in the maturation of tRNA precursors that do not contain an encoded CCA determine. Since all of the 86 E. coli tRNA genes contain an encoded CCA determinant, we sought to determine what the normal substrates were for the E. coli RNase Z homologue. Our experiments have shown that RNase Z serves as a backup enzyme in the initiation of mRNA decay (Perwez & Kushner, 2006). The second new enzyme is called RNase LS (Otsuka & Yonesaki, 2005). This enzyme also seems to be involved as a backup in mRNA decay. Furthermore, it has recently been shown that E. coli contains a pyrophosphate dehydrolase (RppH) that converts 5 terminal triphosphate into a 5 phosphomonoester (Deana et al., 2008) (Fig. 7). With the identification of these enzymes, there are now six characterized endoribonucleases (RNase E, RNase G, RNase III, RNase P, RNase Z and RNase LS) that play some role in the processing and decay of RNA molecules in E. coli. A working model for mRNA decay involving all seven enzymes is shown in Fig. 7. In order to test this model, we are currently using high density tiling arrays that contain both strands of the E. coli genome, at 20 nt resolution, to examine changes in the steady-state levels of various transcripts in the presence or absence of particular enzymes. Our initial experiments have involved comparing the transcriptome profiles of wild type, rne?1018 and rnc-14 strains. This approach has allowed us to not only examine the fate of mRNAs but also to identify potential new sRNAs. We are in the processing of expanding this analysis to include other individual ribonucleases as well as multiple mutants. |
 |
Fig. 6. Endonucleolytic initiation of mRNA decay for a polycistronic transcript. Intercistronic regions are marked by small black vertical bars. Since RNase E, the most abundant endoribonuclease is inhibited by the presence of a 5 triphosphate, the first step in the decay of many mRNAs is the action of RppH to convert the 5 triphosphate to a 5 phosphomonoester. Subsequently, RNase E, will bind to the 5 phosphomonoester terminus to initiate decay. Its binding sterically prevents the binding of RNase LS at a contiguous site. However, the ability of RNase III to cleave the stem-loop structure in the intercistronic region is independent of RNase E action. Similarly, RNase P cleavage within the downstream intercistronic region is also independent of the initial RNase E cleavage. In addition, the downstream RNase G and RNase Z cleavage sites may be recognized, independent of RNase E binding at the 5 terminus, if there are sufficient amounts of each enzyme present. Thus the first round of endonucleolytic cleavage events could yield from between 5-7 decay intermediates. Subsequent cleavages by RNase E, RNase LS, RNase G and RNase Z could lead to a total of 11 decay intermediates if all of the sites are cleaved. It is possible, that some cleavages will not take place, if exonucleolytic degradation of the initial decay intermediates proceeds so rapidly that some endonucleolytic cleavage sites are actually degraded before they are recognized by their respective enzymes. In addition, it should also be noted that Baker and Mackie ( 2003) have shown that under certain circumstances RNase E can cleave at internal sites without binding to a 5 terminus. Sizes of the various endonucleases reflect an estimate of their relative participation in mRNA decay. For the sake of simplicity, RNase E is shown without the other components of the degradosome. The products of the endonucleolytic cleavages will subsequently be degraded by a combination of PNPase, RNase II, RNase R and poly(A) polymerase. |
6. Identification of the primary exonucleases involved in mRNA decay |
| In E. coli, all of the riboexonucleases that have been identified degrade RNA molecules from the 3 terminus. These 3 ? 5 exonucleases fall into two classes depending on their mechanism of catalysis. Hydrolytic enzymes such as RNase II and RNase R release nucleoside monophosphates, while enzymes such as polynucleotide phosphorylase and RNase PH use a phosphorolytic mechanism that requires the presence of inorganic phosphate and releases nucleoside diphosphates. Polynucleotide phosphorylase is a reversible enzyme. We have already shown that PNPase is the primary enzyme involved in the degradation of poly(A) tails associated with mRNAs (Mohanty & Kushner, 2000b) and that it functions both degradatively and biosynthetically in E. coli (Mohanty & Kushner, 2000a). Genome-wide analysis using RNase II and PNPase deletion mutants has demonstrated that PNPase plays a greater role in mRNA decay than RNase II (Mohanty & Kushner, 2003). An unexpected observation from this work was the large percentage of E. coli mRNAs that are destabilized in the absence of RNase II (Mohanty & Kushner, 2003). The recent observation that RNase R is required for the degradation of mRNAs contained large stem-loop structures called repetitive extragenic elements (Cheng & Deutscher, 2005), has led us to construct a series of mutants containing a combination of mutations in PNPase, RNase II and RNase R so that we can gain a better understanding of how these three exonucleases function in mRNA decay. |
References |
| Apirion, D. & A. B. Lasser, (1978) A conditional
lethal mutant of Escherichia coli which affects the processing of
ribosomal RNA. Journal of Biological Chemistry 253: 1738-1742.
Arraiano, C. M., S. D. Yancey & S. R. Kushner, (1988) Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia coli K-12. Journal of Bacteriology 170: 4625-4633. Babitzke, P., L. Granger & S. R. Kushner, (1993) Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III. Journal of Bacteriology 175: 229-239. Baker, K. E. & G. A. Mackie, (2003) Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli. Molecular Microbiol. 47: 75-88. Callaghan, A. J., M. J. Marcaida, J. A. Stead, K. J. McDowall, W. G. Scott & B. F. Luisi, (2005) Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover. Nature 437: 1187-1191. Cheng, Z.-F. & M. P. Deutscher, (2005) An important role for RNase R in mRNA decay. Molecular Cell 17: 313-318. Chung, D.-H., Z. Min, B.-C. Wang & S. R. Kushner, (2009) Single amino acid changes in the predicted RNAse H domain of E. coli RNase G lead to the complementation of RNase E mutants. RNA: submitted. Deana, A., H. Celesnik & J. G. Belasco, (2008) The bacterial enzyme RppH triggers messenger RNA degradation by 5' pyrophosphate removal. Nature 451: 355-358. Granger, L. L., E. B. O'Hara, R.-F. Wang, F. V. Meffen, K. Armstrong, S. D. Yancey, P. Babitzke & S. R. Kushner, (1998) The E. coli mrsC gene is required for cell growth and mRNA decay. Journal of Bacteriology 180: 1920-1928. Kushner, S. R., (2007) Messenger RNA decay. In: Escherichia coli and Salmonella: cellular and molecular biology. A. Böck, R. Curtis III., C. A. Gross, J. B. Kaper, F. C. Neidhardt, T. Nyström, K. E. Rudd & S. C. L. (eds). Washington, DC: American Society for Microbiology Press, pp. http://www.ecosal.org. Li, Z. & M. P. Deutscher, (2002) RNase E plays an essential role in the maturation of Escherichia coli tRNA precursors. RNA 8: 97-109. Mohanty, B. K. & S. R. Kushner, (1999) Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. Mol. Microbiol. 34: 1094-1108. Mohanty, B. K. & S. R. Kushner, (2000a) Polynucleotide phosphorylase functions both as a 3' - 5' exonuclease and a poly(A) polymerase in Escherichia coli. Proc. Natl. Acad. Sci. USA 97: 11966-11971. Mohanty, B. K. & S. R. Kushner, (2000b) Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli. Mol. Microbiol. 36: 982-994. Mohanty, B. K. & S. R. Kushner, (2002) Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E. Molecular Microbiol. 45: 1315-1324. Mohanty, B. K. & S. R. Kushner, (2003) Genomic analysis in Escherichia coli demonstrates differential roles for polynucleotide phosphorylase and RNase II in mRNA abundance and decay. Molecular Microbiol. 50: 645-658. Mohanty, B. K. & S. R. Kushner, (2006) The majority of E. coli mRNAs undergo post-transcriptional modification in exponentially growing cells. Nucleic Acids Research 34: 5695-5704. Mohanty, B. K. & S. R. Kushner, (2007) Ribonuclease P processes polycistronic tRNA transcripts in Escherichia coli independent of ribonuclease E. Nucleic Acids Research 35: 7614-7625. Mohanty, B. K. & S. R. Kushner, (2008) Rho-independent transcription terminators inhibit RNase P processing of the secG leuU and metT tRNA polycistronic transcripts in Escherichia coli. Nucleic Acids Research 36: 364-375. Mohanty, B. K., V. F. Maples & S. R. Kushner, (2004) The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. Molecular Microbiol. 54: 905-920. O'Hara, E. B., J. A. Chekanova, C. A. Ingle, Z. R. Kushner, E. Peters & S. R. Kushner, (1995) Polyadenylylation helps regulate mRNA decay in Escherichia coli. Proc. Natl. Acad. Sci. USA 92: 1807-1811. Otsuka, Y. & T. Yonesaki, (2005) A novel endoribonuclease, RNase LS, in Escherichia coli. Genetics 169: 13-20. Ow, M. C. & S. R. Kushner, (2002) Initiation of tRNA maturation by RNase E is essential for cell viability in Escherichia coli. Genes and Development 16: 1102-1115. Ow, M. C., Q. Liu & S. R. Kushner, (2000) Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly. Molecular Microbiol. 38: 854-866. Ow, M. C., Q. Liu, B. K. Mohanty, M. E. Andrew, V. F. Maples & S. R. Kushner, (2002) RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters. Molecular Microbiol. 43: 159-171. Ow, M. C., T. Perwez & S. R. Kushner, (2003) RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue, RNase E. Molecular Microbiol. 49: 607-622. Perwez, T., D. Hami, V. F. Maples, Z. Min, B.-C. Wang & S. R. Kushner, (2008) Intragenic suppressors of temperature sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli. Nucleic Acids Research 36: 5301-5318. Perwez, T. & S. R. Kushner, (2006) RNase Z in Escherichia coli plays a significant role in mRNA decay. Molecular Microbiology 60: 723-737. Schierling, K., S. Rosch, R. Rupprecht, S. Schiffer & A. Marchfelder, (2002) tRNA 3' end maturation in archaea has eukaryotic features:the RNase Z from Haloferax volcanii. J. Molecular Biology 316. Schiffer, S., S. Rosch & A. Marchfelder, (2002) Assigning a function to a conserved group of proteins: the tRNA 3'processing enzymes. EMBO Journal 21: 2769-2677. |