Trm140 has two recognition modes for 3-methylcytidine modification of the anticodon loop of tRNA substrates.

The 3-methylcytidine (mC) modification is ubiquitous in eukaryotic tRNA, widely found at C in the anticodon loop of tRNA, tRNA, and some tRNA species, as well as in the variable loop (V-loop) of certain tRNA species. In the yeast , formation of mC requires Trm140 for six tRNA substrates, including three tRNA species and three tRNA species, whereas in , two Trm140 homologs are used, one for tRNA and one for tRNA The occurrence of a single Trm140 homolog is conserved broadly among Ascomycota, whereas multiple Trm140-related homologs are found in metazoans and other fungi. We investigate here how Trm140 protein recognizes its six tRNA substrates.

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae.

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA(His) guanylyltransferase accumulate uncharged tRNA(His) lacking the G(-1) residue and subsequently accumulate additional 5-methylcytidine (m(5)C) at residues C(48) and C(50) of tRNA(His), due to the activity of the m(5)C-methyltransferase Trm4.

The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase.

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs-the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p-interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3.

The yeast rapid tRNA decay pathway primarily monitors the structural integrity of the acceptor and T-stems of mature tRNA.

tRNAs, like other RNAs, are subject to quality control steps during and after biosynthesis. We previously described a rapid tRNA degradation (RTD) pathway in which the 5'-3' exonucleases Rat1 and Xrn1 degrade mature tRNA(Val(AAC)) in yeast mutants lacking m(7)G and m(5)C, and mature tRNA(Ser(CGA)) in mutants lacking Um and ac(4)C. To understand how the RTD pathway selects substrate tRNAs among different tRNAs lacking the same modifications, we used a genetic screen to examine tRNA(Ser(CGA)) variants.

A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop.

The 3-methylcytidine (m³C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA(Thr(IGU)) from abp140-Δ strains lacks m³C.

The spindle positioning protein Kar9p interacts with the sumoylation machinery in Saccharomyces cerevisiae.

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele.

The CLIP-170 orthologue Bik1p and positioning the mitotic spindle in yeast.

Bik1p is the yeast Saccharomyces cerevisiae representative of the CLIP-170 family of microtubule plus-end tracking proteins. Bik1p shares a number of similarities with its mammalian counterpart CLIP-170, including an important role in dynein function. However, Bik1p and CLIP-170 differ in several significant ways, including the mechanisms utilized to track microtubule plus ends.

The CLIP-170 homologue Bik1p promotes the phosphorylation and asymmetric localization of Kar9p.

Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p.