Nucleolar localization of early tRNA processing

Abstract

There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis. Received May 21, 1998; revised version accepted June 23, 1998. The physical organization of transcription and RNA processing events in eukaryotic nuclei has been studied extensively in the cases of pre-rRNAs and pre-mRNAs. Different stages in the expression of these RNAs are often associated with distinctive subnuclear structures, and these associations are integral to the synthesis and processing events. There is no direct information on the sites of tRNA gene transcription, although it is generally assumed that tRNA synthesis will be distributed throughout the nucleus; unlike the tandemly repeated ribosomal genes, the tRNA genes are not usually clustered in the chromosomes. In the budding yeast Saccharomyces cerevisiae, the 275 tRNA genes are scattered throughout the linear genome map (Cherry et al. 1997). Information on the sites of the numerous pre-tRNA processing events in the nucleus is also limited and has not provided a clear model for localization of the pathway.Maturation of eukaryotic pre-tRNA primary transcripts is often an ordered process, with 5Ј-and 3Ј-terminal processing preceding splicing (Tobian et al. 1985;Lee et al. 1991) and intron removal in turn preceding exit from nucleus to cytoplasm. In addition, multiple nucleotide modifications occur in the nucleus (Etcheverry et al. 1979;Hopper and Martin 1992). Although the order of processing events is not uniform for all pre-tRNAs and is not absolutely obligatory even for individual pre-tRNAs, the ordering suggests possible spatial organization of the maturation pathway. In yeast, current information on the sites of pre-tRNA processing could be consistent with either a dispersed pathway or one that is highly localized, especially to some portion of the nuclear periphery. Immunoelectron microscopy showed at least some of the tRNA splicing ligase to be near nuclear pores, leading to the speculation that the late splicing events in the nuclear pathway occur near the time that intron-containing pre-tRNAs exit to the cytoplasm (Clark and Abelson 1987). For intron-containing pretRNAs, it is therefore assumed that most of the nuclear precursor will retain the introns and that earlier processing events, such as removal of the termini, will need to take the introns into account (Leontis et al. 1988). Immunofluorescence microscopy has also localized a base modification enzyme, N 2 ,N 2 -dimethylguanosine-s...

Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes

“…In wild-type cells, pre-tRNAs are concentrated at the nucleolus (Fig. 3), as expected (Bertrand et al 1998;L. Wang et al 2005).…”

“…This DNase treatment consistently removed the association of pol III with condensin but left the condensin signal in TFIIIB and TFIIIC pulldowns essentially un- Condensin mutants show loss of tRNA gene and pre-tRNA nucleolar localization. Fluorescence in situ hybridization to the tRNA Leu genes and the early precursor tRNAs (pre-tRNA Leu ) in wild-type cells shows the genes and their transcripts clustered at the nucleolus, as described previously (Bertrand et al 1998;Kendall et al 2000;Thompson et al 2003;L. Wang et al 2005).…”

“…As confirmation of mislocalized pre-tRNA synthesis in strains where the genes are too dispersed to give focal signals, the locations of pre-tRNA Leu (CAA) primary transcripts were also probed (Bertrand et al 1998;L. Wang et al 2005).…”

Functional Proteomic Analysis of Human Nucleolus

“…Therefore, this study, by allowing the classification of a total of ϳ350 nucleolar proteins according to the biological processes in which they are involved, firmly confirms the plurifunctional nature of nucleoli and outlines biological processes taking place within these nuclear domains (Andersen et al, 2002;this study). In particular, the present analysis shows clearly that translational regulators, chaperones, and also proteins involved in mRNA processing are found within nucleoli, in addition to other components of the translation machinery such as ribosomes (Leary and Huang, 2001), tRNAs (Bertrand et al, 1998;Pederson and Politz, 2000), signal recognition particle ; this study), and even mRNAs (Kalland et al, 1991;Bond and Wold, 1993;Kadowaki et al, 1994a). This provides molecular evidence to the recent demonstration that translation can occur within nuclei of human cells (Iborra et al, 2001) and suggests that nucleoli themselves could play a central role in the control of this process.…”

Identification of factors that promote biogenesis of tRNACGASer

“…The mechanisms by which Mot1, Rpa49 and Rrn3 influence tRNA biogenesis is unclear, but it is possible that they indirectly influence the process through their involvement in Pol I transcription. Transcription of tRNA genes as well as 5´-end processing of pre-tRNAs has been reported to localize to the nucleolus [47–49]. Moreover, dissociation of pre-tRNA from the nucleolus and defects in 5´ end processing have been observed in cells defective in Pol I transcription or rRNA processing [50,51].…”