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CCA That Is Also Transcribed. the Sequence Surrounding the Site Of promoters, and a 3’-CCA that is also transcribed. The sequence surrounding the site of catalysis for RNase P, at the 5’ mature end of the tRNA, is quite different from one RNA species to another. In contrast to the tRNA sequence diversity, the structure of all bacterial tRNAs is quite similar. The commonly portrayed secondary structure of tRNAs resembles a cloverleaf, with four stems: the acceptor , T!C, anticodon, and D arms (Figure 1.1). Long-range nucleotide interactions define the 3-dimensional shape of tRNAs with two helices made up of co-axial arrangements of the D and anticodon stems and the T!C and aminoacyl stems (Figure 1.2). There is some degree of structural variability in tRNAs, as the “variable loop” (between the anticodon and D arms) can vary in length. Consequently, B. subtilis tRNAs vary in length from 70-92 nucleotides (nt). The minimal substrate structure for RNase P cleavage is the coaxial stems consisting of the acceptor stem and the T-stem with the T!C-loop [1]. However, bacterial RNase P recognition is also affected by several other tRNA structures, including the D-, anticodon and variable arms, as well as the 5’ leader sequence and a Watson-Crick interaction between the 3’-CCA and an internal loop in the bacterial RNase P RNA subunit [8-12]. In addition to pre-tRNAs, bacterial RNase P is known to process several substrates that are proposed to contain tRNA-like structures. These bacterial substrates include 4.5S RNA, tmRNA, viral RNAs, mRNAs, riboswitches, ColE1 replication origin control RNAs, and C4 antisense RNA from phages P1 and P7 [13-21]. The presence of the 2 Figure 1.1. Representative secondary structure of a pre-tRNA The cloverleaf secondary structure is one of the defining factors of a tRNA. Shown here is yeast pre-tRNAPhe. The variable loop exists between the Anticodon and T!C arms. 3 Figure 1.2. Three dimensional structure of a pre-tRNA The cloverleaf structure illustrates the Watson-Crick basepair interactions, however the three-dimensional folding of a pre-tRNA also involves long range nucleotide interactions to form the co-axial stem that makes up the minimal substrate for RNase P. 4 protein subunit in the RNase P holoenzyme increases the substrate versatility of the enzyme over the RNA enzyme alone [7]. Eukaryotic RNase P The function of nuclear RNase P in eukaryotic systems is much more complicated and less understood. First, there are two very similar enzymes in the nucleus that are related to bacterial RNase P, named RNase P and RNase MRP. Yeast RNase P is known to be responsible for processing pre-tRNAs, and RNase MRP is needed for the processing of a pre-rRNA (5.8S) and the regulated turnover of a cell cycle mRNA [22, 23]. Second, both RNase P and RNase MRP from nuclei are far more complex enzymes than bacterial RNase P [24]. Each enzyme still employs a distinctive, but related, RNA subunit and contains multiple protein subunits required for function in vivo. Yeast RNase P contains nine protein subunits: Rpr2p, Rpp1p*, Pop1p*, Pop3p*, Pop4p*, Pop5p*, Pop6p*, Pop7p*, Pop8p* [25]. Yeast RNase MRP, while a physically distinct enzyme, also contains the protein subunits with asterisks next to them as well as Snm1p and Rmp1p [26-29]. The eukaryotic RNase P RNA subunit alone is not generally considered to be catalytically active, although recent experiments have demonstrated the RNA subunit might contain a remnant of catalytic activity with an observed catalytic rate five to six orders of magnitude lower than the bacterial RNA-only enzyme [30]. This suggests that the eukaryotic enzyme relies heavily on its protein constituent. One known role for the 5 Figure 1.3. Comparison of RNase P holoenzyme from bacteria and yeast RNase P from eukaryotes is a much more complex enzyme. While the RNAs are approximately the same size in bacteria and eukaryotes, the protein content changes dramatically. The bacterial RNase P protein is 10% (by mass) of the holoenzyme. In yeast, proteins make up 70% of the mass of the holoenzyme. Given the bacterial proteins role in substrate binding, specifically with the non-tRNA substrates, this massive increase in protein might facilitate the binding of multiple types of RNA substrates for cleavage by the catalytic RNA subunit. 6 7 eukaryotic RNase P proteins is to ensure proper folding of the RNA subunit [31], but other functions remain unclear. Yeast RNase P substrates Although even the simple bacterial enzyme is known to cleave multiple non-pre-tRNA substrates, little is known about the diversity of substrates for eukaryotic RNase P. The huge increase in the protein content of the holoenzyme compared to the bacterial enzyme suggests the potential for a large increase in the variety of substrates that may be recognized. This would be akin to the increased diversity of promoters recognized by the structurally complex eukaryotic RNA polymerases, compared to the bacterial RNA polymerase, which contains a similar catalytic core. In yeast, the only defined set of RNase P substrates is the pre-tRNAs; no additional non-pre-tRNA substrates have yet been identified. In mammalian systems, even the set of pre-tRNA substrates has never previously been defined. There are 274 tRNA genes in the yeast genome, encoding a set of pre-tRNAs that are more diverse than in bacteria. In addition to the diversity of tRNA sequence, in yeast the 3’-CCA sequences are not encoded as they are in bacteria, and some yeast tRNAs also contain introns. Introns are found in 61 yeast tRNA genes, corresponding to nine yeast tRNA families: Ile1, Leu1, Leu3, Lys2, Phe, Pro1, Ser3, Ser5, Trp, Tyr. The size of yeast introns ranges from 14-60nt long. In most cases RNase P removes the 5’ leader before the intron and 3’ trailing sequences are removed, indicating RNase P must be able to accommodate both intron-containing and intron-less tRNAs. Indeed, it has been 8 suggested that the presence of introns in some tRNAs facilitates their correct tertiary folds, allowing increased primary sequence diversity without compromising recognition by processing enzymes that depend on tertiary structure [32]. Although no physiological non-pre-tRNA substrate has yet been identified, a recent report has indicated that the non-coding RNA HRA1 is an in vitro substrate for the glycerol gradient fraction that includes RNase P [33]. Another recent study used microarray analysis to examine the RNAs affected when Rpp1p, one of the subunits common to both RNase P and RNase MRP, is depleted. There was an effect on the general mRNA population, but no specific RNA substrates were determined [34]. The authors did identify 74 transcripts, all from intergenic and antisense regions of the genome, that accumulate with Rpp1p depletion suggesting either RNases P or MRP might regulate these RNAs. Multiple characteristics of the eukaryotic enzyme suggest that nuclear RNase P has additional substrates. First, seven of the nine protein subunits in yeast nuclear RNase P are highly positively charged (pI 9.3-10.0), which could provide multiple binding sites for negatively charged RNAs. This is consistent with previous studies on substrate binding kinetics that suggest eukaryotic RNase P has at least two RNA binding sites [35]. Second, eukaryotic RNase P is 1,000-times more susceptible to inhibition by single stranded homoribopolymers than bacterial RNase P [35], suggesting that the holoenzyme strongly binds single stranded RNAs in ways that inhibit pre-tRNA recognition. Experiments have shown potent, sequence-specific inhibition of yeast nuclear RNase P 9 by poly-U and poly-G RNAs, even greater than inhibition by pre-tRNAs (Ki <10 nM for poly-A and poly-G, compared to >20 nM for pre-tRNA) [35]. This demonstrates RNase P’s ability to bind single stranded RNAs is at least as good as its ability to bind pre- tRNAs. Finally, a temperature sensitive mutation (S827S829) in one of the shared subunits between RNase P and RNase MRP, Pop1p, leads to cell death without affecting either pre-tRNA or 5.8S rRNA processing [36]. This indicates that the mutation has a lethal effect on an as yet undefined target. These three points, in addition to the precedence of the multiple substrates of bacterial RNase P and yeast RNase MRP, strongly suggests the possibility of non-tRNA substrates for eukaryotic RNase P. The work reported in chapter II details our search for additional tRNA substrates for the nuclear form of yeast RNase P. First, we examine which RNAs physically associate with RNase P by identifying RNAs that copurify with the enzyme, using whole genome microarray analysis. We then ask which RNAs increase in abundance in yeast strains with mutations in different subunits of RNase P, also utilizing microarrays. Finally, we use northern blot analysis to examine multiple potential substrates for processing defects in the temperature sensitive mutant strains. Through this comprehensive approach, we identify numerous mRNAs that are both physically associated with RNase P and accumulate in the mutant strains. Furthermore, we identify the family of box C/D intron- encoded small nucleolar RNAs (snoRNAs) as physically associating with RNase P and accumulating a processing intermediate due to RNase P mutations. Focused examination of this group of snoRNAs has shown that RNase P is likely to participate in this processing pathway. 10 Mammalian RNase P substrates In mammalian systems, it is surprisingly not known which tRNAs are actually expressed. There simply is no comprehensive analysis of expressed tRNAs of the type that has been performed for yeast. Presently, there are only 11 mouse tRNA sequences verified as expressed by direct sequencing of tRNAs [37]. Although there is a database of tRNA genes predicted by tRNAscan-SE available for all completed genomes [38], so far there has been little confirmation of the predictive power of tRNA scanning programs in the mammalian genome.
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