Research

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 cell’s 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 (Kushner, 2007). 

Interestingly, the processing and decay of noncoding RNAs is carried out by the same enzymes that are involved in mRNA decay.  Since most noncoding RNAs (tRNAs, rRNAs) are synthesized as much larger primary transcripts that have to undergo extensive processing before they become functionally active, many of the enzymes involved in the processing and decay of mRNAs are also involved in these processing steps.  Furthermore, there are a number of ribonucleases whose functions are uniquely related to the maturation of both rRNAs and tRNAs.  Many of these same enzymes also play important roles in the biogenesis and degradation of small regulatory RNAs (sRNAs), which have recently been shown to play important roles in the post-transcriptional regulation of a large number of mRNAs. 

Another fascinating aspect of RNAs in E. coli is the post-transcriptional addition of poly(A) tails by poly(A) polymerase I (Mohanty and Kushner, 1999) and polynucleotide tails by polynucleotide phosphorylase (Mohanty and Kushner, 2000).  Experiments from our laboratory (Mohanty and Kushner, 1999; O'Hara et al., 1995) have demonstrated that poly(A) tails can specifically serve as a targeting mechanism for the rapid degradation of mRNAs.  However, our very recent work has revealed an important new function for polyadenylation.  Specifically, we have shown that polyadenylation is involved in regulating functional levels of tRNAs (Mohanty et al., 2012) and that deregulation of poly(A) polymerase I leads to the rapid inhibition of protein synthesis, which causes cell death (Mohanty and Kushner, 2013).

           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 understand the function and molecular mechanism of polyadenylation in E. coli.  Previous experiments in our laboratory have shown that poly(A) tails not only 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 and Kushner, 1999, 2002).  We have also shown that poly(A) polymerase I is part of a multiprotein complex that 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 and Kushner, 2006; Mohanty et al., 2004).  A genomic analysis of the entire E. coli transcriptome has shown that almost 90% of the E. coli ORFs undergo some degree of polyadenylation (Mohanty and Kushner, 2006). 

However, many questions remain to be answered.  For example, the analysis of polyadenylation is complicated by the fact that while poly(A) polymerase accounts for 90% of the poly(A) tails in E. coli (Mohanty and Kushner, 1999), polynucleotide phosphorylase (PNPase) also synthesizes polynucleotide tails in wild-type bacteria (Mohanty and Kushner, 2000; Mohanty et al., 2004).  However, 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.   Interestingly, more prokaryotes contain PNPase rather than a true poly(A) polymerase. 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, RNase Z, 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 either PNPase dissociates or the substrate is processed into a small oligonucleotide (4-7 nt) that is degraded by oligoribonuclease, the product of the orn gene. 

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 either as monocistronic genes or polycistronic loci that contain multiple tRNAs, tRNAs and rRNAs, or tRNAs and mRNAs.  A variety of data have suggested that RNase E is responsible for the initial separation of all polycistronic transcripts that contain tRNA precursors (Li and Deutscher, 2002; Ow and Kushner, 2002) (Fig. 2).

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.  Model is based on data  from Li and Deutscher (Li and Deutscher, 2002) and Ow and Kushner (Ow and Kushner, 2002).            

While this model explains the initial processing of many primary tRNA transcripts, 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 and Kushner, 2007).  In the case of valV valW (Fig. 3), the initial cleavages by RNase P generate pre-tRNAs with mature 5' termini after the Rho-dependent terminator has been removed by a combination of RNase II and PNPase. The processing at the 3' termini is similar to that described in Fig. 2.  We have also shown that the secG leuU and metT operons also employ RNase P to separate the tRNAs (Mohanty and 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 and Kushner, 2008).  Interestingly, not all primary tRNA transcripts utilize either RNase E or RNase P for their initial processing steps.   In the case of the monocistronic leuX transcript, the Rho-independent transcription terminator is removed primarily by the 3' ® 5' exonucleolytic activity of PNPase (Mohanty and Kushner, 2010).  This result was quite surprising, since PNPase has been shown to be inhibited by secondary structures (Spickler and Mackie, 2000).  However, PNPase does not degrade all of the downstream sequences.  The final 3' end processing is carried out by either RNase T or RNase PH (Mohanty and Kushner, 2010).

We are currently examining the processing of additional primary tRNA transcripts that do not seem to employ any of the enzymatic pathways described above.  For example, it is not clear how the monocistronic proK and proL transcripts have their Rho-independent transcription terminators removed.

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. Interface between polyadenylation, tRNA processing and protein synthesis

During our analysis of tRNA processing, we occasionally observed that a small percentage of pre-tRNAs contained very short poly(A) tails (1-5 nt) downstream of their immature 3' termini  (Mohanty and Kushner, 2008).  Upon more careful analysis, we determined that in the absence of RNase T and RNase PH, most pre-tRNAs (79/86) were subject to polyadenylation (Mohanty et al., 2012).  Subsequently, we showed that PAP I, RNase T and RNase PH compete for pre-tRNAs, helping to regulate functional tRNA levels (Fig. 4) (Mohanty et al., 2012).

Figure 4.  Model for the interaction of RNase T, RNase PH and PAP I.  RNase E cleaves the polycistronic argX operon to generate individual pre-tRNAs, followed by RNase P cleavages that generate the mature 5' ends on the pre-tRNAs.  The mechanism for removal of the transcription terminator following proM is unkown (double question marks).  The long 3' trailer sequences associated with some pre-tRNAs that are generated by RNase E cleavage are often trimmed backe to 2-3 nt downstream of the CCA determinant by RNase II.  In wild type cells the resulting pre-tRNAs are rapidly processed by a combination of RNase T and RNase PH to generate mature tRNAs that are aminoacylated.  A small percentage of pre-tRNAs are polyadenylated by PAP I.  These polyadenylated species are slowly converted to mature forms by a currently unknown mechanism (double question marks).  In the absence of RNase T, RNase PH or both enzymes, the fraction of polyadenylated pre-tRNAs increases significantly, resulting in substantially slower growth rates in these mutants (Mohanty et al., 2012).

Subsequently, we have investigated an observation that we made in 1999 (Mohanty and Kushner, 1999), in which deregulation of PAP I synthesis led to a loss of cell viability.  It turns out that increased synthesis of PAP I leads to the rapid polyadenylation of mature tRNAs, resulting in an inhibition of protein synthesis and subsequent cell death (Mohanty and Kushner, 2012).  Interestingly, unlike poly(A) tails that are found on mRNAs, the polyadenylation of tRNAs only produces short tails (<10 nt) even in the presence of increased levels of PAP I (Mohanty et al., 2012). 

Based on these findings, we believe that polyadenylation in E. coli is far more prevalent than previously thought.  We are currently trying to determine why a small subset (7) of the tRNA transcripts are not substrates for PAP I.

4. Analysis of the role of RNase E in mRNA decay

RNase E (Fig. 5), 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 and Lasser, 1978).  Subsequently, it was shown to be involved in mRNA decay (Arraiano et al., 1988) and the maturation of tRNAs (Ow and Kushner, 2002).  Since RNase E 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 and Kushner, 2002).  Of further interest is the fact that RNase E serves as the scaffold for a multiprotein complex called the "degradosome" (Fig. 5).   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 and Kushner, 2002). 

Additional experiments have shown that the rne gene is controlled by a complex regulatory system that involves three distinct promoters (Ow and Kushner, 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. 5).  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. 

Figure 5. 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. (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. (Perwez et al., 2008).  

5. Examine the relationship between polyadenylation and the RNase E-based degeadosome  

We have recently observed an apparent linkage between polyadenylation of mRNAs containing Rho-independent transcription terminators and the presence of a functional RNase E-based degradosome.  RNase E is composed of a N-terminal catalytic region and a C-terminal scaffold region (Vanzo et al., 1998).  The E. coli degradosome contains RNase E, PNPase, the RhlB RNA helicase and enolase (Carpousis et al., 1994; Py et al., 1996).  It appears that in the absence of the scaffold region, PAP I does not recognize Rho-independent transcription terminators.  We are currently trying to determine the link between polyadenylation and the RNase E-based degradosome.

6. 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. 6).  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 effectively process tRNA precursors (Ow et al., 2003).   Recently, we have isolated single-amino acid substitution mutations in the RNase G protein that lead to its ability to support cell viability in RNase E deletion mutants (Chung et al., 2010).  Interestingly, domain swaps between RNase G and RNase E failed to generate any functional proteins (Chung et al., 2010).  We are currently studying the mutant RNase G proteins at the biochemical level to determine how its catalytic activities have been altered.

Fig. 6.  Comparison  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. (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., 2010).

7. 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 decay for specific mRNAs (Arraiano et al., 1988; Babitzke et al., 1993; Granger et al., 1998; O'Hara et al., 1995; 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), RNase Z has been shown to be involved in the maturation of tRNA precursors that do not contain an encoded CCA determinant.  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 homolog.  Our experiments have shown that RNase Z serves as a backup enzyme in the initiation of mRNA decay (Perwez and Kushner, 2006). The second new enzyme is called RNase LS (Otsuka and Yonesaki, 2005).  This enzyme also seems to be involved as a backup in mRNA decay.  

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, rneD1018 and rnc-14 strains.  This approach has allowed us to not only examine the fate of mRNAs but also to identify potential new small regulatory RNAs (sRNAs) (Stead et al., 2010).  We are in the processing of expanding this analysis to include other individual ribonucleases as well as multiple mutants.

Fig. 7.  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 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, and RNase R. Decay intermediates containing secondary structures will be polyadenylated by poly(A) polymerase I to promote their degradation by PNPase, RNase II, or RNase R.

8.  Analysis of the 5' end decapping in mRNA decay and tRNA processing 

In eukaryotes, the 5' termini of mRNAs are post-transcriptionally modified by the addition of a methyl-G cap.  This structure protects the mRNA from 5' ® 3' decay and promotes translation.  In prokaryotes, the 5' ends of all primary transcripts contain a triphosphate.  However, RNase E, the major endoribonuclease in E. coli, is inhibited by the presence of a triphosphate (Mackie, 2000).  In fact, Deanna and Belasco (Deana et al., 2008) discovered that the “nudix” protein RppH encodes a pyrophospohydrolase that effectively converts the 5' triphosphate into a 5' monophosphate.  Using a microarray, they examined how the failure to remove 5' triphosphates affected the stray-state levels of all E. coli ORFs (Deana et al., 2008).  Since all primary tRNA transcripts have 5' triphosphates, we are currently examining if RppH plays a role in tRNA processing.

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