RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

tRNA 3' end maturation in archaea has eukaryotic features: the RNase Z from Haloferax volcanii

Here, we report the first characterization and partial purification of an archaeal tRNA 3' processing activity, the RNase Z from Haloferax volcanii. The activity identified here is an endonuclease, which cleaves tRNA precursors 3' to the discriminator. Thus tRNA 3' processing in archaea resembles the eukaryotic 3' processing pathway. The archaeal RNase Z has a KCl optimum at 5mM, which is in contrast to the intracellular KCl concentration being as high as 4M KCl.The archaeal RNase Z does process 5' extended and intron-containing pretRNAs but with a much lower efficiency than 5' matured, intronless pretRNAs. At least in vitro there is thus no defined order for 5' and 3' processing and splicing. A heterologous precursor tRNA is cleaved efficiently by the archaeal RNase Z. Experiments with precursors containing mutated tRNAs revealed that removal of the anticodon arm reduces cleavage efficiency only slightly, while removal of D and T arm reduces processing effciency drastically, even down to complete inhibition. Comparison with its nuclear and mitochondrial homologs revealed that the substrate specificity of the archaeal RNase Z is narrower than that of the nuclear RNase Z but broader than that of the mitochondrial RNase Z.     

Alteration of a mitochondrial tRNA precursor 5'' leader abolishes its cleavage by yeast mitochondrial RNase P.

A mitochondrial specific RNase P is required to process 5' leaders from mitochondrial tRNA precursors in Saccharomyces cerevisiae. Experiments with a pair of mitochondrial pretRNAs(Asp) having leaders of different base composition suggest that this enzyme is unexpectedly sensitive to leader sequence or structure. Asp-AU (75% AU leader) is cleaved by the mitochondrial RNase P while Asp-GC (39% AU) is not. Both are substrates for E. coli RNase P. Partial nuclease digestions show that the tRNA portions of the two precursors differ in tertiary structure, while their 5' leaders differ in secondary structure. It is unusual for an RNaseP to have substrate specificity requirements which preclude processing of a pretRNA known to be a suitable substrate for an RNaseP from another species.     

Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.     

Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5[prime] leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRNAArgs containing a 13-nt 3' trailer and a 5[prime] leader of various lengths. The 3' processing was slightly stimulated by 5[prime] leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths. We also investigated whether 3'tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem. Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.     

Mammalian 2',3' cyclic nucleotide phosphodiesterase (CNP) can function as a tRNA splicing enzyme in vivo

Yeast and plant tRNA splicing entails discrete healing and sealing steps catalyzed by a tRNA ligase that converts the 2',3' cyclic phosphate and 5'-OH termini of the broken tRNA exons to 3'-OH/2'-PO4 and 5'-PO4 ends, respectively, then joins the ends to yield a 2'-PO4, 3'-5' phosphodiester splice junction. The junction 2'-PO4 is removed by a tRNA phosphotransferase, Tpt1. Animal cells have two potential tRNA repair pathways: a yeast-like system plus a distinctive mechanism, also present in archaea, in which the 2',3' cyclic phosphate and 5'-OH termini are ligated directly. Here we report that a mammalian 2',3' cyclic nucleotide phosphodiesterase (CNP) can perform the essential 3' end-healing steps of tRNA splicing in yeast and thereby complement growth of strains bearing lethal or temperature-sensitive mutations in the tRNA ligase 3' end-healing domain. Although this is the first evidence of an RNA processing function in vivo for the mammalian CNP protein, it seems unlikely that the yeast-like pathway is responsible for animal tRNA splicing, insofar as neither CNP nor Tpt1 is essential in mice.     

tRNA核酸内切酶的研究进展

tRNA在蛋白质合成过程中起着极其重要的作用。在所有的生物体内,tRNA首先以前体形式转录,然后必需经过一系列的加工后才能成为有功能的tRNA分子。tRNaseZ、RNaseP和tRNA剪接内切酶是参与tRNA前体加工的三种主要的核酸内切酶,分别参与tRNA前体3′末端、tRNA前体5′末端和内含子剪接的加工。这三种酶具有不同的结构特征,并且利用完全不同的催化机制水解磷酸二酯键。tRNaseZ和RNaseP都是金属酶,活性中心分别需要Zn^2＋和Mg^2＋的参与;而tRNA剪接内切酶活性中心不需要金属离子,是一个由不同催化亚基上的关键氨基酸残基构成的组合式活性中心。