The best-characterized substrates for La binding are pre-tRNAs, and La binding to these and other pol III transcripts is dependent of the length of the 3' terminal oligo(U) tract. Human La protein binds best to RNAs that contain 3 or more terminal Us [14
], whereas a direct comparison indicates that S. pombe
La clearly requires 4 or more terminal Us for efficient binding [78
]. As the S. pombe
pol III requires at least 5T residues as a minimal terminator whereas human pol III requires only 4 [79
], these co-variations suggest that binding of nascent pol III RNAs by La protein is functionally linked to the pol III termination system. Moreover, transcription termination by pol III on a particular gene produces a heterogeneous set of transcripts that differ in the length of their 3' oligo-U tracts. Typically, the most abundant RNA species end with one less rU residue than the minimal number of dT residues required for termination, and as alluded to above, it is this nascent RNA species that can be efficiently engaged by La protein. However a substantial fraction of the nascent pol III transcripts from the same gene ends with fewer U residues than required for efficient La binding [15
]. By following the phenotype of a functional suppressor-tRNA in S. pombe
, Huang et al. observed that this 3' end heterogeneity leads nascent transcripts that differ in their La binding to two different posttranscriptional tRNA maturation pathways [78
]. Moreover, the extent to which the pol III subunit, Rpc11p, executes its 3' exonuclease activity during the transcription termination process can be a determinant of terminal oligo-U length and therefore the degree to which the nascent transcripts are engaged by La [78
] (). One consequence of this heterogeneity is that the La-associated pre-tRNAs and the La-independent pre-tRNAs are differentially sensitive to the 3'-5' exosome-TRAMP nuclear surveillance system [80
] (see below).
Scheme depicting the role of the 3' end binding activity of La protein in different pathways of precursor-tRNA processing in yeast
Studies in budding and fission yeast indicate that in the absence of La most pre-tRNAs follow a variant tRNA processing pathway (). During pre-tRNA processing, La binds and protects the 3’ ends of pre-tRNAs from at least two exonucleases, the 3’ exonuclease Rex1p (pathway 1 in ) and for defective pre-tRNAs, the nuclear exosome-Rrp6 (pathway 4 in ) [80
]. After processing of the 5’ leader by RNAse P, the 3' end-protected trailer is processed by the endonuclease RNAse Z (pathways 1 & 3 in ) precisely after the discrimator base, the first unpaired nucleotide following the acceptor stem [82
], leading to dissociation of La from the tRNA and addition of CCA to the new 3' end [87
]. In the absence of La, and for those pre-tRNAs that do not engage La even when it is present [88
] (pathway 2 in ), the order of pre-tRNA processing may be altered such that exonucleolytic trimming of 3’ trailers precedes 5’ leader cleavage by RNAse P [13
Yet, precisely how La and other factors contribute to the 3’ end metabolism of all pre-tRNAs in the cell is not fully clear. For example, as noted above although La is required for normal 3’ endonucleolytic processing of many pre-tRNAs in S. cerevisiae
], some pre-tRNAs do not use the La-dependent pathway even when it is present [88
]. Rex1p [81
] is a 3' riboexonuclease that functionally processes pre-tRNAs in the absence of La and presumably also those pre-tRNAs that undergo 3' exonucleolytic processing even when La is present. For those pre-tRNAs that do use it, La appears to promote endonucleolytic processing by blocking access of the 3’ ends to the processing exonucleases, i.e., Rex1p [81
]. Accordingly, since La is nonessential in yeast, and in its absence, pre-tRNAs gain access to Rex1p for productive processing, it might have been expected that in cells lacking La, the 3’ endonuclease, RNAse Z, would be nonessential. Intriguingly, this is not the case in fission yeast, S. pombe
]. It remains to be determined if this is also true in S. cerevisiae
and also if it reflects the possibility that RNAse Z may be required for an essential yeast function other than pre-tRNA processing..
It has been shown that La deletion is synthetically lethal with yeast mutations that disrupt the secondary structure of essential tRNAs [18
]. This leads to susceptibility of the structurally-impaired pre-tRNA to degradation, in this case, by a second class of 3’ exonuclease distinct from Rex1p, the surveillance activities of the TRAMP polyadenylation complex and the nuclear exosome which degrades aberrant RNAs [93
]. Furthermore, ectopic La can complement the deletion of tRNA modification enzymes that catalyze addition of modifications that presumably stabilize tRNA structure [93
]. The absence of proper modification can sensitize some pre-tRNAs to degradation by the surveillance exosome, which can be averted by the 3’ end binding activity of La [93
]. In the case of pre-tRNAiMet, failure to be methylated on A58 by the tRNA methyltransferase TRM6-TRM61, leads to nuclear degradation of the hypomodified precursor tRNA; remarkably however, overexpression of La can avert this degradation, permitting the hypomethylated but otherwise mature tRNAiMet to persist and be exported to the cytoplasm where it apparently functions in translation [93
]. This latter observation suggests that a critical function of methylation of A58 of tRNAiMet is to avoid decay of the pre-tRNAiMet in the nucleus.
Some functions of La in pre-tRNA processing reflect a La activity beyond simple 3’end protection [80
]. Several lines of evidence indicate that La exhibits a complex function related to RNA structure/folding. In addition to the capacity of La to complement the loss of tRNA modifications thought to stabilize tRNA structure, La mutants have been described which harbor wild-type 3’ end binding and protection activity but are still incapable of rescuing the maturation of certain structurally impaired pre-tRNAs, even when Rrp6, a 3' exonuclease component of the nuclear surveillance exosome, is deleted [80
]. These results are consistent with functions for La in pre-tRNA processing that are more complex than simple UUU-3’OH binding and protection from the 3’ surveillance exonucleases [80
]. Indeed, the 3' end binding activity and the second, more complex activity which allows La to function in the rescue of structurally-impaired pre-tRNAs, have been localized to the two different RNA binding surfaces of the LAM and RRM1 of the human and S. pombe
La proteins [80
Attempts to reconcile simple vs. complex functions in RNA metabolism have become a critical theme in the study of the mechanisms that control La function: simple functions related to UUU-3’OH mediated RNA binding and 3’ end protection, vs. complex La functions that involve enhancing the folding and/or stability of various RNA targets via poorly understood binding mode(s) and mechanisms. Although this has begun to be addressed in the context of pre-tRNA processing [80
], understanding precisely how the coordinated use of the separate RNA binding surfaces of the LAM and RRM1 contribute to other activities such as in mRNA-related functions, during RNA chaperone activity, and for La binding to its more stably associated pol III transcripts such as cellular Y RNAs, as well as viral encoded EBER and VA1 RNAs remains a challenge.
As described in detail below, recent crystallographic and biochemical studies have advanced understanding of the variations of La-RNA binding modes and associated functions significantly. Another advance related to these outstanding issues involves the increasingly studied LARPs [21
]. Just as genuine La proteins combine various mechanisms of RNA binding to provide complex functions, it would appear that an emerging theme in LARP function might also involve variations on similar modes to provide distinct functions on their own sets of RNA targets. The diversity of function of La proteins and LARPs, and their expected shared and divergent manners of RNA recognition, will be the topic of the remainder of this review article.