An intrinsically disordered C terminus allows the La protein to assist the biogenesis of diverse noncoding RNA precursors

Abstract

The La protein binds the 3′ ends of many newly synthesized noncoding RNAs, protecting these RNAs from nucleases and influencing folding, maturation, and ribonucleoprotein assembly. Although 3′ end binding by La involves the N-terminal La domain and adjacent RNA recognition motif (RRM), the mechanisms by which La stabilizes diverse RNAs from nucleases and assists subsequent events in their biogenesis are unknown. Here we report that a conserved feature of La proteins, an intrinsically disordered C terminus, is required for the accumulation of certain noncoding RNA precursors and for the role of the Saccharomyces cerevisiae La protein Lhp1p in assisting formation of correctly folded pre-tRNA anticodon stems in vivo. Footprinting experiments using purified Lhp1p reveal that the C terminus is required to protect a pre-tRNA anticodon stem from chemical modification. Although the C terminus of Lhp1p is hypersensitive to proteases in vitro, it becomes protease-resistant upon binding pre-tRNAs, U6 RNA, or pre-5S rRNA. Thus, while high affinity binding to 3′ ends requires the La domain and RRM, a conformationally flexible C terminus allows La to interact productively with a diversity of noncoding RNA precursors. We propose that intrinsically disordered domains adjacent to well characterized RNA-binding motifs in other promiscuous RNA-binding proteins may similarly contribute to the ability of these proteins to influence the cellular fates of multiple distinct RNA targets.RNA-binding protein | unstructured domain | noncoding RNA biogenesis M any RNA-binding proteins interact with multiple ligands, influencing the fate of the RNAs and in some cases altering structure. For example, the pre-mRNA binding protein hnRNP A1 and the mRNA decay mediator KSRP also bind miRNA precursors to promote their maturation (1, 2). Similarly, the Y-box protein YB-1, a component of cytoplasmic mRNPs, functions in both pre-mRNA splicing and translational repression of some mRNAs (3). Although most of these proteins contain well characterized RNA-binding motifs, the mechanisms by which they interact with diverse targets and influence their conformations and outcomes are not understood.One such protein, called La, is a nuclear phosphoprotein whose best characterized role is to assist noncoding RNA biogenesis. La binds the UUU OH that is the initial end of all RNA polymerase III transcripts and protects the RNAs from exonucleases (4, 5). In budding yeast, La also binds processing intermediates of RNA polymerase II-transcribed small nuclear and nucleolar RNAs that end in UUU OH (6-8). Because most La-bound RNAs undergo 3′ maturation, La is usually not bound to the mature RNAs. Binding by La results in diverse consequences. For pretRNAs, La binding favors endonucleolytic removal of the trailer (9) and is required for wild-type levels of some tRNAs (10). For the yeast U4 snRNA, the La-bound precursor preferentially associates with the core Sm proteins in vitro, suggesting that La assists snRNP formation by protecting the favored substrate for Sm prot...

xRRM

“…18 Yeast genuine La (Lhp1p) does not have a C-terminal RRM2 domain; however, it has a C-terminal unstructured extension after RRM1 that is required for several Lhp1p functions. 1,31 This region of the protein gets structured and protease resistant upon binding to certain structured noncoding RNA precursors, including pretRNA. 31 Based on these observations, it Figure 6.…”

“…1,31 This region of the protein gets structured and protease resistant upon binding to certain structured noncoding RNA precursors, including pretRNA. 31 Based on these observations, it Figure 6. Model of assembly of Tetrahymena telomerase catalytic core.…”

“…The region C-terminal to the La module in genuine La and LARP7 proteins has been implicated in RNA chaperone and/ or folding functions. 13,[29][30][31] This part of the protein contains what we have identified as the xRRM2 (Fig. 1A).…”

“…1A). 13,31 How the xRRM2 interacts with RNA and whether its RNA folding functions are associated with the La module in genuine La and LARP7 proteins in general is not clear. The C-terminal extension includes important functional elements in hLa protein, such as the nuclear localization signal (NLS), a short basic motif (SBM), a nuclear retention element (NRE) and several phosphorylation sites.…”

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

“…Recent years have witnessed a tremendous proliferation of three-dimensional structure determinations by the advent of high throughput x-ray crystallography soon after the sequence and adequate expression of a protein became available (1,2). In some proteins, no electron density can be observed for stretches of the amino acid sequence especially at the N or C terminus (3)(4)(5)(6)(7)(8). Thus, a functional role for such regions is not always discernible from their three-dimensional structures.…”