Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide‐specific and intron‐dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre‐tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(‐) pus1(‐) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

The loop of a stem structure close to the 5 end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A 1 , the 5 end of the 18S rRNA, and at site A 2 , located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3-pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5 external transcribed spacer (5 ETS). Cleavage at site A 0 in the yeast 5 ETS strictly requires base pairing between U3 and a sequence within the 5 ETS. In contrast, the U3-18S interaction is not required for A 0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A 1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A 1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.Eukaryotic nucleoli contain a large number of small nucleolar RNA (snoRNA) species, most of which function as guides for rRNA modifications. However, a small number of snoRNAs are required for processing of the pre-rRNA (reviewed in references 21 and 36), of which the most studied is U3. Genetic depletion of U3 in the yeast Saccharomyces cerevisiae inhibits three early pre-rRNA cleavage reactions on the pathway of 18S rRNA synthesis (Fig. 1); cleavage is inhibited at sites A 0 (in the 5Ј external transcribed spacer [5Ј ETS]), A 1 (the 5Ј end of the mature 18S rRNA), and A 2 (in internal transcribed spacer 1 [ITS1]) (14). In contrast, the cleavage of site A 3 and sites further in the 3Ј direction on the pathway of 5.8S and 25S synthesis is unaffected by depletion of U3. Depletion of the U3-associated proteins Nop1p, Sof1p, and Mpp10p leads to essentially identical phenotypes (8,15,37), indicating that the intact U3 small nucleolar ribonucleoprotein (snoRNP) particle is required for pre-rRNA cleavage at these sites. Depletion of U3 has also been reported to inhibit in vitro cleavage of the mouse 5Ј ETS (16) and pre-rRNA processing in Xenopus oocytes (5, 30).In vivo psoralen cross-linking experiments identified several sites of interaction between the yeast U3 snoRNA and the pre-rRNA. One was a single-stranded region in the 5Ј region of the U3 snoRNA (nucleotides [nt] 39 to 48) which exhibited a 10-nt complementarity to a region of the 5Ј ETS (nt 470 to 479; approximately 140 nt 5Ј to site A 0 and 230 nt 5Ј to site A 1 ) (3, 4). Disruption of this base pairing blocked cleavage at sites A 0 , A 1 , and A 2 and accumulation ...

We have screened nucleoporin mutants for the inhibition of tRNA splicing, which has previously been proposed to be coupled to transport. Strains mutant for Nup49p or Nup116p, or genetically depleted of Nup145p, strongly accumulated unspliced pre-tRNAs. Splicing was inhibited for all 10 families of intron-containing pre-tRNA, but no effects on 5' or 3' end processing were detected. Strains mutant for Nup133p or Nsp1p accumulated lower levels of several unspliced pre-tRNAs. In contrast, no accumulation of any pre-tRNA was observed in strains mutant for Nup1p, Nup85p, or Nup100p. Other RNA processing reactions tested, pre-rRNA processing, pre-mRNA splicing, and small nucleolar and small nuclear RNA synthesis, were not clearly affected for any nucleoporin mutant. These data provide evidence for a coupling between pre-tRNA splicing and nuclear-cytoplasmic transport. Mutation of NUP49, NUP116, or NUP145 has previously been shown to lead to nuclear poly(A)+ RNA accumulation, indicating that these nucleoporins play roles in the transport of more than one class of RNA.

No abstract

In yeast, the 59 end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A 1 , the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 59 region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A 1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A 1 . There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A 1 and A 2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease.