Yeast Messenger RNA Research Articles

Identification of Inosine and 2'-O-Methylinosine Modifications in Yeast Messenger RNA by Liquid Chromatography-Tandem Mass Spectrometry Analysis.

The discovery of reversible modifications in messenger RNA (mRNA) opens new research directions in RNA modification-mediated epigenetic regulation. Yeast is an extensively used model organism in molecular biology. Systematic investigation and profiling of modifications in yeast mRNA would promote our understanding of the physiological regulation mechanisms in yeast. However, due to the high abundance of ribosomal RNA (rRNA) and transfer RNA (tRNA) in total RNA, isolation of low abundance of mRNA frequently suffers from the contamination of rRNA and tRNA, which will lead to the false-positive determination and inaccurate quantification of modifications in mRNA. Therefore, obtaining high-purity mRNA is critical for precise determination and accurate quantification of modifications in mRNA, especially for studies that focus on discovering new ones. Herein, we proposed a successive orthogonal isolation method by combining polyT-based purification and agarose gel electrophoresis purification for extracting high-purity mRNA. With the extracted high-purity yeast mRNA, we systemically explored the modifications in yeast mRNA by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The results showed that in addition to the previously reported eight kinds of modifications, two novel modifications of inosine (Ino) and 2'-O-methylinosine (Im) were identified to be prevalent in yeast mRNA. It is worth noting that Im was reported for the first time, to the best of our knowledge, to exist in living organisms in the three domains of life. Moreover, we observed that the levels of 10 kinds of modifications including Ino and Im in yeast mRNA exhibited dynamic change at different growth stages of yeast cells. Furthermore, Im in mRNA showed a significant decrease while in response to H2O2 treatment. These results indicated that the two newly identified modifications in yeast mRNA were involved in yeast cell growth and response to environmental stress. Taken together, we reported two new modifications of Ino and Im in yeast mRNA, which expends the diversity of RNA modifications in yeast and also suggests new regulators for modulating yeast physiological functions.

Structural similarity in the absence of sequence homology of the messenger RNA export factors Mtr2 and p15.

The association between Mtr2 and Mex67 is essential for the nuclear export of bulk messenger RNA in yeast. In metazoans, the analogous function is carried out by the TAP-p15 heterodimer. Whereas Mex67 and TAP are highly conserved proteins, their binding partners, Mtr2 and p15, share no sequence similarity, but are nevertheless functionally homologous. Here, we report the 2.8-A resolution crystal structure of Mtr2 in complex with the NTF2-like domain of Mex67. Mtr2 is a novel member of the NTF2-like family and interacts with Mex67, forming a complex with a similar structural architecture to that of TAP-p15. Mtr2 fulfils an analogous function to that of human p15 in maintaining the structural integrity of the heterodimer. In addition, Mtr2 presents a long internal loop, which contains residues that affect the export of the large ribosomal subunit.

Myosin-V: head to tail.

The myosin-V family is the most extensively studied of the unconventional myosin families. Most organisms examined have at least one member of the myosin-V family: many have multiple members. The wide range of species in which myosin-V has been identified suggests that myosin-V is a fundamental component of organelle transport in all higher eukaryotes. Possible cargoes for myosin-V range from melanosomes and synaptic vesicles in mammals to vacuoles and messenger RNA in yeast. In this review, we discuss the current state of research on the cellular function of myosin-V as described by the actions of the head, neck and tail domains.

Structural basis for the regulation of splicing of a yeast messenger RNA

In S. cerevisiae, ribosomal protein L32 regulates the splicing of the transcript of its own gene, RPL32. We have identified an RNA structure within the transcript that is responsible for this regulation. Initial deletions limited essential sequences to the 5′ exon and the first few nucleotides of the intron. To take advantage of phylogenetic comparison of RNA structures, RPL32 was cloned from the closely related species, Kluyveromyces lactis. The splicing of Its transcript is similarly regulated. Sequences conserved between the S. cerevisiae and K. lactis transcripts suggested a structure involving base pairing of a region encompassing the 5′ splice site with another nearthe 5′ end of the transcript. Analysis of numerous site-directed mutations supports this structure. We infer that stabilization of this structure by L32 inhibits splicing by precluding the interaction of U1 RNA with the 5′ splice site.

Minimal methylation of yeast messenger RNA

Most, if not all, yeast mRNAs are capped at their 5′-terminus by m7G. Apart from m7G no other methylated nucleotides could be detected in poly (A)+ mRNA isolated from yeast polysomes.

Methylated nucleosides in polyadenylate-containing yeast messenger ribonucleic acid

Yeast messenger RNA was found to be methylated. A calculation of the specific methylation showed that the average yeast messenger RNA molecule contains only two methylated nucleosides which occur in one alkali stable oligonucleotide. Similar to other eukaryotic messengers, the 5′ terminus of yeast messenger RNA is blocked by 7-methylguanosine, linked through a di- or triphosphate bridge to a ribosemethylated nucleoside. Contrary to the messengers of higher eucaryotic organisms, no additional base methylated constituents were found.

Half life of yeast messenger RNA.

WE have determined the half life of Saccharomyces cerevisiae messenger RNA (mRNA) and its associated poly (A) sequences, from the kinetics of their labelling with 14C-adenine. Corrections have been made for changes in the ATP pool specific activity during the labelling period. This method of determining the mRNA half life is preferred to observing the decay of mRNA in the presence of inhibitors because changes in the cell metabolism caused by an inhibitor may also change the mRNA half life1. One of our purposes in determining these half lives is for their use in the characterisation of temperature-sensitive mutants which are defective in macromolecular synthesis2. For this reason we have determined the half lives of mRNA and poly(A) in two different conditions, at 23 and 36 °C.

Turnover of polyadenylated messenger RNA in fission yeast. Evidence for the control of protein synthesis at the translational level.

Polyadenylated RNA was isolated from fission yeast (Schizosaccharomyces pombe) total RNA using oligo(dT)-cellulose, and was studied as a model for messenger RNA. The half-life of poly adenylated RNA was measured by two independent methods. (a) The rate of labelling of polyadenylated RNA during incubation of cells with [5-3H]uridine was measured. A half-life of 40-45 min was found by comparing the experimental data with theoretical curves calculated for labelling of RNAs with various half-lives. The influence of precursor-pool specific activity on RNA labelling kinetics is considered. (b) Cells were labelled with [5-3H]uridine then further RNA synthesis was inhibited by addition of 8-hydroxyquinoline. The rate of loos of radioactivity from polyadenylated RNA indicated a half-life of 50 min. The half-life found by these two methods is about one-third of the cell doubling time, and is much longer than previous estimates by indirect methods of yeast messenger RNA half-life. Both experimental methods provided evidence for the existence of tas a half-life of 40-50 min; a much smaller population is probably turning over more rapidly. After inhibition of RNA synthesis by 8-hydroxyquinoline, the rate of total protein synthesis declined much more rapidly than the polyadenylated RNA content of the cells. However, 60 min after inhibition of RNA synthesis there was a small rise in the rate of portein synthesis. These data are interpreted as evidence for mechanisms controlling protein synthesis which operate at the level of messenger RNA translation.

Biogenesis of polysomes and transport of messenger RNA in yeast.

The study of polysomal formation in the ts-136 thermo-sensitive yeast mutant indicates that ribosomes are assembled with an mRNA, which is in a structure tightly bound to membranes, and are then released into the soluble cytoplasmic fraction. This has been observed by studying the recruitment of ribosomes when glucose is added to glucose-starved cells and when transport of mRNA is permitted by shifting the mutant to the permissive temperature. A soluble cytoplasm and a fraction becoming soluble after sodium deoxycholate treatment of a rapidly sedimenting structure have been characterized. The former contains the majority of polysomes, free 80-S monomers and almost all of the ribosomal subunits. The latter fraction is composed of bound 80-S monomers and polysomes, but lacks ribosomal subunits. Treatment of the rapidly sedimenting structure with pancreatic ribonuclease produces the release of 80-S monomers, with EDTA the release of an equal proportion of both ribosomal subunits, and with sodium deoxycholate the release of 80-S monomers and polysomes. These findings are consistent with the assumption that bound ribosomes are assembled with an mRNA which is tightly bound to this rapidly sedimenting structure, presumably membranes. From the operational viewpoint this fraction is called the "membrane." During the process of polysomal formation ribosomes are recruited more rapidly in the "membranes" than in the soluble cytoplasm. Since "membranes" do not accumulate polysomes and contain only a small fraction of the total amount of ribosomes, the result is consistent with the assumption that either there is a higher turnover of bound versus free polysomes or bound polysomes are the precursors of free polysomes. The latter assumption is more likely since we have shown previously (a) that in yeast, transport is coupled with the translation of bound mRNA, (b) that this mRNA is tightly bound to a structure which sediments very rapidly and becomes soluble only after sodium deoxycholate treatment, and (c) when cycloheximide is added during the recruitment of ribosomes there is accumulation of membrane-bound ribosomes.

Translation of poly(riboadenylic acid)-enriched messenger RNAs from the yeast, Saccharomyces cerevisiae, in heterologous cell-free systems.

Poly(riboadenylic acid) [poly(A)] enriched messenger RNAs from yeast have been used to direct the synthesis of yeast polypeptides in mouse Krebs II ascites and wheat embryo extracts. Both cell-free systems, synthesize polypeptides over a molecular weight range of 10,000-100,000. Autoradiograms of sodium dodecyl sulfate-polyacrylamide slab gels used to fractionate [35S]methionine-labeled in vitro products reveal that about 25 major bands (each of them possibly representing multiple polypeptides) are produced by each cell-free system. Each of these coelectrophoreses with a major polypeptide labeled in vivo or in a yeast lysate. These results suggest that cell-free translational machinery from eukaryotes is not able to discriminate in an all or none fashion against messenger RNAs which are available to it. While yeast poly(A)-enriched messenger RNA directs the synthesis polypeptides over approximately the same molecular weight range in both cell-free systems, the wheat germ system directs the incorporation of 45 times the amount of [3H]serine into Cl3CCOOH-precipitable polypeptides. This is in contrast to the 2.5-fold more efficient translation of hemoglobin mRNA in the wheat embryo extract. Thus, the extract from mammalian cells is able to translate mRNA from a lower plant with a much lower efficiency than it translates hemoglobin mRNA, and at a lower efficiency than is observed using a cell-free system from wheat embryos. This indicates that the wheat embryo system is the one of choice for translation of yeast messenger RNA.

The polyadenylate polymerases from yeast.

Poly(A) polymerase activity was first detected in yeast extracts, primarily in association with the ribosomal fraction, by Twu and Bretthauer in 1971 (Twu, J. S., and Bretthauer, RK. (1971) Biochemistry 10, 1576-1582). This activity has now been separated into three distinct enzymes by chromatography on DEAE-cellulose. Each of the three enzymes can catalyze the incorporation of adenylate residues from ATP into a polyadenylate (poly(A)) tract at the 3' terminus of a primer RNA. Enzyme I elutes at 0.07 M ammonium sulfate from the DEAE-cellulose column, utilizes the mixed polynucleotide poly(A,G,C,U) or ribosomal RNA most efficiently in vitro, and may be responsible in vivo for the initiation of the poly(A) tracts found on yeast messenger RNA. Enzyme II elutes from the column at 0.20 M ammonium sulfate, requires poly(A) itself or an RNA primer containing a 3'-oligo(A) tract, and may be responsible in the nucleus for the elongation of tracts initiated by enzyme I. Enzyme III elutes from the column at 0.56 M ammonium sulfate and is present in low amounts in nuclear extracts. It may be involved in adding poly(A) tracts to messenger RNA in mitochondria. These enzymes also have the intrinsic capacity for the incorporation of cytidylate residues from CTP, which correlates with the finding of cytidylate residues in the poly(A) tracts present in the yeast RNA which is rapidly labeled in vivo. About 75% of the total poly(A) polymerase activity of yeast is enzyme I, most of which is present in the soluble protein fraction of the whole yeast extract. About 20% of the total poly(A) polymerase is enzyme II, and 1 to 5% is enzyme III. All three of the yeast poly(A) polymerases require an RNA primer with a free 3'-hydroxyl group, show no requirement for a DNA template, require Mn-2+ for optimal activity, have pH optima of 8.5, and are inhibited by GTP, CTP, UTP, and native yeast DNA. Polymerases I and II have similar molecular weights by gel filtration.

Monocistronic messenger RNA in yeast

We have determined the rate of polypeptide chain synthesis on different size polysomes in yeast. The completion time for the average polypeptide chain in vivo at 23 °C is two minutes by this technique and is in good agreement with values we have determined by other independent methods. These kinetic experiments indicate that the average size of a nascent polypeptide chain on a polysome is directly related to the size of the polysome. This demonstrates that in the simple eucaryotic organism, Saccharomyces cerevisiae, mRNA is monocistronic in the sense that each mRNA molecule codes for one protein molecule which is released intact from the ribosome upon completion. The pattern of amino acid incorporation into Escherichia coli polysomes is distinctly different. These findings have a number of interesting implications for the genetics of the lower eucaryotes and indicate that the cellular mechanisms of control and co-ordination in yeast may differ from those found in procaryotes and may be similar to cellular mechanisms of control for mammalian cells.