Conserved and divergent features of the structure and function of La and La-related proteins (LARPs)

Abstract

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3'OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3'OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3'OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

Figure 1

A) Schematic alignment indicating the conserved LAM-RRM domain architecture of the genuine La proteins of three species (top, S. cerevisiae Lhp1p, S. pombe Sla1p, and human hLa) followed by LARPs, arranged in order of their sequence conservation in their LAMs (see B below). Numbered RRM-L3, RRM-L4, RRM-L5 motifs reflect variations of these from the canonical RRM and their conservation of these across LARP families [1]. Note that while the RRM of LARP4 is routinely predicted as such by the NCBI BLAST server, the RRMs of LARPs 1 and 6 were not (unpublished observations) but were found using special methods [1]. Three characterized members of the LARP7 family are shown, Tth: Tetrahymena thermophila LARP-p65; Eu: Eupliotes aediculatus LARP-p43, and hLARP7, also known as PIP7S [21, 143, 150]. The LARPs 6, 1, & 4 families are represented by their human members, along with coexisting subfamily members hLARPs 1b and 4b (see text). Features indicated in the hLa schematic and described in the text include a nuclear export element in RRM1 (E; [112]), a nuclear retention element (R; [110]), a short basic motif (B; [73]), the CKII phosphorylation site S366 (S; [160]) and a nuclear import sequence (N; [108]). B) The RRM1 of La provides two surfaces for pre-tRNA binding: i) at the interface between the RRM and the LAM [76], responsible for UUU-3’OH dependent interaction (RNA corresponding to U-3 to U-1 shown in red), and ii) RRM1 loop-3 (shown in blue), which is important for UUU-3’OH independent binding to pre-tRNAs and possibly other La targets [87]. The D33 side chain (in the LAM) which makes bidentate contacts to the 2' and 3' OH groups of the terminal U (U-1) of the RNA, is shown in blue. RRM1 β-sheet surface residues Y114 and F155, whose side chains are indicated in blue, also function in pre-tRNA processing [80]. Also depicted are two hydrogen bonds that connect the LAM and an underside edge of RRM1, in part through La Y23, and in part through the U-2 base of the RNA.

Figure 1

A) Schematic alignment indicating the conserved LAM-RRM domain architecture of the genuine La proteins of three species (top, S. cerevisiae Lhp1p, S. pombe Sla1p, and human hLa) followed by LARPs, arranged in order of their sequence conservation in their LAMs (see B below). Numbered RRM-L3, RRM-L4, RRM-L5 motifs reflect variations of these from the canonical RRM and their conservation of these across LARP families [1]. Note that while the RRM of LARP4 is routinely predicted as such by the NCBI BLAST server, the RRMs of LARPs 1 and 6 were not (unpublished observations) but were found using special methods [1]. Three characterized members of the LARP7 family are shown, Tth: Tetrahymena thermophila LARP-p65; Eu: Eupliotes aediculatus LARP-p43, and hLARP7, also known as PIP7S [21, 143, 150]. The LARPs 6, 1, & 4 families are represented by their human members, along with coexisting subfamily members hLARPs 1b and 4b (see text). Features indicated in the hLa schematic and described in the text include a nuclear export element in RRM1 (E; [112]), a nuclear retention element (R; [110]), a short basic motif (B; [73]), the CKII phosphorylation site S366 (S; [160]) and a nuclear import sequence (N; [108]). B) The RRM1 of La provides two surfaces for pre-tRNA binding: i) at the interface between the RRM and the LAM [76], responsible for UUU-3’OH dependent interaction (RNA corresponding to U-3 to U-1 shown in red), and ii) RRM1 loop-3 (shown in blue), which is important for UUU-3’OH independent binding to pre-tRNAs and possibly other La targets [87]. The D33 side chain (in the LAM) which makes bidentate contacts to the 2' and 3' OH groups of the terminal U (U-1) of the RNA, is shown in blue. RRM1 β-sheet surface residues Y114 and F155, whose side chains are indicated in blue, also function in pre-tRNA processing [80]. Also depicted are two hydrogen bonds that connect the LAM and an underside edge of RRM1, in part through La Y23, and in part through the U-2 base of the RNA.

Figure 2. Scheme depicting the role of the 3' end binding activity of La protein in different pathways of precursor-tRNA processing in yeast

La is nonessential in yeast but its absence affects the processing of most precursor-tRNAs [12, 13]. La protein is depicted as a colored oval. The degree to which pre-tRNAs require La for maturation depends on their structural integrity. Four pathways are depicted labeled 1–4 under the diagonal arrows leading into the pathways. Most pre-tRNAs probably go through pathway 1. Some pre-tRNAs do not functionally engage La even when present [88], and these would presumably use pathway 2, a La-independent pathway. Pathway 4 reflects the nuclear surveillance pathway for pre-tRNAs [94]. Defective pre-tRNAs are structurally impaired due to mutations that disrupt basepairs in the anticodon stem or variable arm, or lack of appropriate modifications (see text). A curved dashed red line depicts a separate decay pathway due to absence of La activity 2 which is required for the maturation of structurally impaired pre-tRNAs that are degraded even in the absence of Rrp6p [80] (see text); *La activity 2 is mediated by the conical RNA β-surface (and loop-3) of RRM1. Additional red dashed lines depict connectivity and crosstalk between the pathways (see [81, 90]).

Figure 3

A) CLUSTAL-W alignment of the LAM sequences of various La proteins and LARPs. The residues involved in UUU-OH recognition by hLa [76, 77] are indicated by asterisks above and numbering according to hLa, below (see text). B) High resolution structure derived from [77], to depict conservation of residues involved in the UUU-3’OH recognition RNA binding pocket. Crystallographic assignment of residues shown to contact the terminal UUU-3’OH by hLa are indicated (hLa structure and numbering [77]), along with the percentage of identity conservation for the noted La proteins (Sla1p, Lhp1p) and LARP family members (hLARP1, hLARP1b, hLARP4, hLARP4b, hLARP6, hLARP7, p43 and p65).

Figure 3

A) CLUSTAL-W alignment of the LAM sequences of various La proteins and LARPs. The residues involved in UUU-OH recognition by hLa [76, 77] are indicated by asterisks above and numbering according to hLa, below (see text). B) High resolution structure derived from [77], to depict conservation of residues involved in the UUU-3’OH recognition RNA binding pocket. Crystallographic assignment of residues shown to contact the terminal UUU-3’OH by hLa are indicated (hLa structure and numbering [77]), along with the percentage of identity conservation for the noted La proteins (Sla1p, Lhp1p) and LARP family members (hLARP1, hLARP1b, hLARP4, hLARP4b, hLARP6, hLARP7, p43 and p65).