Pre-tRNA processing was strongly affected by depletion of the essential Lsm proteins, Lsm2p to Lsm5p and Lsm8p, whereas the absence of Lsm1p, Lsm6p, or Lsm7p resulted in much weaker phenotypes. This is different from the stability of U6, which requires all seven proteins, Lsm2p to Lsm8p (29
), but similar observations have been made for pre-rRNA processing (J. Kufel, J. Beggs, and D. Tollervey, unpublished observations). It may be that the lack of any essential protein prevents Lsm complex formation, while nonessential proteins can partially replace each other and form complexes that retain substantial activity. During U6 synthesis and in mRNA turnover and translation, Lsm complexes have been proposed previously to act as chaperones that modify the structure of RNP complexes, and a bacterial Sm-like protein functions to promote correct RNA-RNA interactions (29
). The absence of Lsm proteins does not inhibit accumulation of mature tRNAs but alters the pattern of processing intermediates. The absence of the yeast La protein, Lhp1, also perturbed pre-tRNA processing without reducing tRNA levels (51
). Accumulation of pre-tRNAs in the lsm
strains closely resembles the processing pattern of mutant pre-tRNACGASer
allele), in which a mutation in the anticodon stem disturbs tRNA structure and renders processing strictly dependent on Lhp1p (26
). Roles in promoting correct formation of RNP complexes would be consistent with the consequences of Lsm protein depletion for pre-tRNA processing.
Defects in pre-tRNA processing were detected prior to growth inhibition, and coprecipitation of pre-tRNAs was seen with TAP-tagged Lsm3p, consistent with direct effects. Lsm proteins affect pre-mRNA splicing and degradation; however, the tRNA processing phenotypes reported here appear to be specific for lsm
mutants, since they were not observed in strains deficient in either mRNA splicing or mRNA degradation. The function of Lsm proteins in pre-tRNA processing is further supported by two-hybrid interactions (13
) that were reported between Lsm8p and the putative RNA helicase Sen1p, which acts as a positive effector of the tRNA splicing endonuclease (10
), and between Lsm2p and Tpt1p, the 2′-phosphotransferase that functions in tRNA splicing (9
). These interactions would be consistent with the Lsm proteins promoting pre-tRNA splicing by aiding the recruitment of splicing cofactors.
We also observed aberrant 3′-extended pre-tRNAs in Lsm-depleted cells, which were substantially longer than the normal PT. These are likely to represent products of transcriptional read-through that would normally have been rapidly degraded in wild-type cells. A role for the essential Lsm proteins in tRNA degradation is supported by the observation of truncated tRNA fragments in depleted strains. It is currently unclear whether these fragments arise from tRNAs that would normally have been degraded in wild-type cells, but without clear intermediates, in which case the detection of degradation intermediates may reflect a loss of nuclease processivity. Alternatively, the absence of a functional Lsm complex may provoke tRNA degradation, perhaps due to problems in RNA folding that would otherwise have been corrected by an Lsm-associated chaperone activity. In other experiments, degradation of the mature rRNAs was also observed (J. Kufel, J. Beggs, and D. Tollervey, unpublished observations), indicating that this phenomenon is not restricted to tRNAs. In the case of mRNAs, the Lsm1p-Lsm7p complex is reported to confer protection against 3′ degradation by the cytoplasmic exosome, a complex of 3′→5′ exonucleases (19
), while promoting 5′ degradation by the 5′→3′ exonuclease Xrn1p (45
). Two-hybrid interactions have been reported between Lsm2p and Lsm8p and the exosome component Mtr3p and between Lsm2p, Lsm4p, Lsm8p, and Xrn1p (13
), whereas proteomic analysis revealed association of Lsm8p with another component of the exosome, Rrp42p (22
). These are consistent with direct association of the Lsm2p-Lsm8p complex with the RNA degradation machinery. Moreover, defects in 5.8S rRNA synthesis seen in strains depleted of any of the essential Lsm proteins are consistent with reduced processivity of both the exosome and the 5′→3′ exonucleases Rat1p and Xrn1p (J. Kufel, J. Beggs, and D. Tollervey, unpublished observations).
An alternative explanation for the pre-tRNA accumulation seen in the Lsm protein-depleted strains might be that the accumulated species are misfolded or otherwise defective pre-tRNAs that would otherwise have been targeted for rapid degradation. We think that this is less likely, since it would imply a substantial level of pre-tRNA degradation in wild-type cells. Moreover, a similar phenotype was not seen in strains lacking components of the exosome complex (unpublished observations).
Pre-tRNAs also undergo extensive covalent nucleotide modification, and a large number of different modified nucleotides have been identified (39
) which play important roles in tRNA function (reviewed in several chapters of reference 18
). At least in some cases there is evidence for interactions among the tRNA modification, splicing, and export pathways. The requirements for Lsm proteins in pre-tRNA localization, tRNA modification, and export have not yet been addressed.
Interactions between Lsm complexes and Lhp1p.
All RNA PolIII PT are believed to associate with La/Lhp1p, via their 3′ poly(U) tracts. La (and presumably Lhp1p) binds to 3′-terminal poly(U) tracts in vitro (43
), but efficient binding to complex RNP substrates in vivo may be more dependent on cofactors. The binding of Lhp1p to large RNA PolIII transcripts, scR1 and the pre-P RNA, was drastically reduced (76- and 18-fold, respectively) by depletion of Lsm3p or Lsm5p for 24 h, with 11- and 5.5-fold reduction after just 8.5 h. The association of Lhp1p with smaller PolIII transcribed RNAs, pre-5S and tRNA PT, was reduced to a lesser extent, with a threefold reduction following Lsm depletion for 24 h. The synthetic lethality of mutations in LSM5
in combination with lhp1-
) supports their functional interaction.
The less efficient association between Lhp1p and RNAs observed in the absence of Lsm3p and Lsm5p was not due to depletion of Lhp1p, the level of which was unaltered, or to nonspecific inhibition of RNA binding, since the precipitation of the U6 snRNA was substantially increased following Lsm depletion. Lhp1p has been proposed previously to “hand on” U6 to the Lsm2p-Lsm8p complex (21
), and in the absence of Lsm proteins, Lhp1p may remain associated with U6 for a longer period.
A correlation was seen between the pre-tRNAs that were coprecipitated with Lsm3p-TAP and the pre-tRNAs that accumulated in the absence of Lsm proteins, consistent with a direct role for the Lsm complex in their processing. However, the low coprecipitation efficiency suggests that the association of Lsm complexes with tRNA precursors is transient. This, and the reduction of Lhp1p binding to its substrates in the absence of Lsm proteins, is consistent with an Lsm complex functioning as a chaperone in the assembly of pre-tRNA/protein complexes. Potential roles would include facilitating the interaction of pre-tRNAs with processing enzymes and cofactors, including Lhp1p, and/or increasing their specific activities.