Effect of capping upon the mRNA properties of satellite tobacco necrosis virus ribonucleic acid.

The mRNA guanyltransferase-mRNA methyltransferases of vaccinia virions can be used to introduce a 5'-terminal m7g(5')pp(5')Apm... capping group onto the RNA of satellite tobacco necrosis virus (STNV RNA) to yield intact capped STNV RNA. Studies with an in vitro system from wheat germ and limiting quantities of capped and uncapped STNV RNA show that the rates and extents of formation of initiation complexes of protein synthesis by intact capped and uncapped STNV RNA are identical, suggesting that 5'-terminal cap groups cannot function in the translation of STNV RNA. Also, the cap analogue pm7G equally inhibits the initiation and the translation of limiting quantities of both capped and uncapped STNV RNA. These contrasting observations suggest that the wheat germ system contains a pm7G sensitive protein and that STNV RNA has a tertiary structure that restricts the function of an added 5'-terminal capping group. This theory is supported by observations that fragmented capped STNV RNA is better at forming initiation complexes than is equally fragmented uncapped STNV RNA.     

Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei.

RNAs that function in mitochondria are typically encoded by the mitochondrial DNA. However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion. It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import. We have identified and characterized mitochondrial precursor tRNAs in T. brucei. The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors. The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with [alpha-32P]CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P. We show that T. brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T. brucei tRNA precursors. Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis. The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.     

U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase).

The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.     

Formation of lariats and circles in self-splicing of the precursor to the large ribosomal RNA of yeast mitochondria

Self-splicing of the precursor to large ribosomal RNA of yeast mitochondria leads not only to circles but also to lariats, structures that have not been observed before as products of self-splicing. Lariats were studied by electron microscopy after hybridization with an RNA complementary to the 3' half of the precursor. This leads to differentiation in at least two classes of lariats that vary in the position of the branch point. In all lariats the tail carries the 3' end, which suggests that a 5' end is used for branch formation with an internal nucleotide. The circles are formed from excised introns. They lack only three nucleotides encoded by mitochondrial DNA along with the 5'-terminal G added in the course of self-splicing. The diverse number of self-splicing products arising in vitro testifies to the considerable reactivity of this intron. The formation of lariats in an RNA catalyzed reaction may have implications for views on the mechanism of splicing of nuclear pre-mRNAs.     

5'' Caps in hnRNA: absence of m32,2,7G and size distribution of capped molecules.

In HeLa cells the "small nuclear" RNA has a cap II 5' structure (8)-- m32,2,7G(5') pppXmpYmp-- where X and Y are 2'0 methylated adenosine and uridine. In contrast hnRNA contains only cap I structures were the 2'0 methylated residue may be any base as was earlier reported for cytoplasmic mRNA (8,9,11). With a clear distinction between the source of these two caps an analysis of the size distribution of capped hnRNA could be performed which revealed over 65% of the capped hnRNA molecules were larger than cytoplasmic mRNA.     

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs. II. Analysis of 7-methylguanosine in mitochondrial and cytosolic aminoacyl-transfer RNAs.

The 7-methylguanosine (m7G) content of two individual mitochondrial tRNAs, labelled in the aminoacyl moiety was assayed by the specific cleavage of the tRNA at this nucleotide followed by electrophoretic analysis to identify the 3'-terminal fragment of the tRNA. Neither Syriam hamster mitochondrial tRNALeu nor tRNAMet were found to contain m7G. In contrast, cytosolic tRNAMetS were cleaved indicating the presence of m7G, apparently 27--28 and 29 nucleotides from their 3' terminus. Cystolic tRNALeu was not cleaved. These results are discussed in relationship to the reported low content of methylated nucleosides in mitochondrial 4 S RNA.     

鲤鱼线粒体tRNA~(phe)基因结构的特异性

鲤鱼线粒体tRNA~(phe)基因的核酸序列已被测定。在鲸、人、爪蟾、牛、小鼠、鸡和鲤鱼中对此基因序列比较发现在D茎存在一个奇怪的保守结构,然而D茎在其余种类的已经测定的脊椎动物线粒体tRNA基因和细胞质tRNA基因中是极不保守的。这一保守结构包含有13bp碱基,我们将此保守区前7个碱基与真核生物RNA PolⅢ识别的A区相比较,发现在此不同物种的两种序列存在部分的同源性。考虑到tRNA~(phe)基因在线粒体基因组上位于置换环区和线粒体rRNA基因编码区之间这一特殊区域内,我们推测这一奇怪的保守结构可能存在其它更为有意义的功能。     

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila.

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.