xRRM: a new class of RRM found in the telomerase La family protein p65

Abstract

Genuine La and La-related proteins group 7 (LARP7) bind to the non-coding RNAs transcribed by RNA polymerase III (RNAPIII), which end in UUU-3'OH. The La motif and RRM1 of these proteins (the La module) cooperate to bind the UUU-3'OH, protecting the RNA from degradation, while other domains may be important for RNA folding or other functions. Among the RNAPIII transcripts is ciliate telomerase RNA (TER). p65, a member of the LARP7 family, is an integral Tetrahymena thermophila telomerase holoenzyme protein required for TER biogenesis and telomerase RNP assembly. p65, together with TER and telomerase reverse transcriptase (TERT), form the Tetrahymena telomerase RNP catalytic core. p65 has an N-terminal domain followed by a La module and a C-terminal domain, which binds to the TER stem 4. We recently showed that the p65 C-terminal domain harbors a cryptic, atypical RRM, which uses a unique mode of single- and double-strand RNA binding and is required for telomerase RNP catalytic core assembly. This domain, which we named xRRM, appears to be present in and unique to genuine La and LARP7 proteins. Here we review the structure of the xRRM, discuss how this domain could recognize diverse substrates of La and LARP7 proteins and discuss the functional implications of the xRRM as an RNP chaperone.

Keywords: LARP7; La protein; RNA conformation; Tetrahymena; p65; telomerase; telomerase RNA; xRRM2.

Figure 1. hLa and LARP7 proteins and Tetrahymena telomerase RNA. (A) Domain architecture of hLa, Tetrahymena p65, Euplotes p43 and human 7SK LARP7. Telomerase LARP7 p65 has a Tetrahymena specific N-terminal domain (in cyan) that is absent in other LARP7 proteins. (B) The secondary structure and conserved elements in Tetrahymena TER: S1 is stem 1, S2 is stem 2, SL4 is stem-loop 4, S4 is stem 4, TBE is template boundary element, PK is pseudoknot and TRE is template recognition element. The secondary structure and nucleotide sequence of the S4 construct used in this study is shown in the inset. The native nucleotides are in blue with numbering from full-length TER and the two (non-conserved) G-C base pairs at the bottom and terminal G(UUCG)C loop at the top are in gray. (C) The structure of p65 xRRM2 domain (PDB ID 4EYT). All the secondary structure elements are marked. The positions of the 46 residue β2-β3 loop and the unstructured C-terminal tail are marked. The RNP3 residues Y407 and D409 on β2, and conserved R465 on β3 are marked. (D) Structure of hLa RRM2 structure (PDB ID 1OWX). All the secondary structure elements are marked. The non-canonical helix α3 is in red. (E) Schematic representation of a canonical RRM with the position of RNP1 and RNP2 shown. (F) Schematic representation of xRRM with position of RNP3, conserved R465 and helix α3x shown. Residues of RNP3 in p65 xRRM2 (Y-X-D) shown.

Figure 2. The p65 xRRM2 and its interaction with RNA. (A) The structure of p65 xRRM2 domain in the RNA-bound state. All the secondary structure elements are the same as in Figure 1C. The C-terminal residues that form a helix upon binding to the double-stranded RNA are marked as α3x. The position of the key RNP3 residues Y407 and D409, and conserved R465 in the bound state are marked. A curved arrow depicts the movement of Y407 side-chain upon stacking on G121 of TER S4. (B and C) Conformational change in TER SL4 upon p65 xRRM2 binding. (B) Free structure of SL4 of Tetrahymena TER (based on PDB ID 2FEY). (C) Model of TER SL4 bound to p65 xRRM2. The model is based on p65 xRRM2 bound to TER S4 (PDB ID 4ERD), the loop 4 is taken from the solution structure (PDB ID 2M21) and placed on the distal stem 4.

Figure 3. Isothermal titration calorimetry (ITC) data and analysis of xRRM2 mutants with TER S4. (A) TER S4 and p65 xRRM2-Y407A mutant (from RNP3). (B) S4 and p65 xRRM2-R465A (conserved R465 in β3). (C) TER S4 and p65 xRRM2(Δ416-456). (D) TER S4 and p65 xRRM2(Δ413-450). The K_Ds of interactions are shown on each panel.

Figure 4. Proposed binding modes for recognition of different RNAs by the xRRM. (A) Cartoon depiction of xRRM, the β-sheet is shown in orange oval and helix 3 (α3+α3x) is shown in red cylinder based on p65 xRRM2. (B) Cartoon depiction of p65 xRRM2 and TER S4 interaction. (C‒E) Different scenarios of single- and double-strand RNA interaction mode for xRRM2 of La and LARP7 protein.

Figure 5. Structure of the hLa RRM1 (PDB ID 2VOP). All the secondary structure elements are marked. RNP1 and RNP2 residues 114 and 155, respectively, and non-canonical helix α3 are marked.

Figure 6. Model of assembly of Tetrahymena telomerase catalytic core. The known and proposed binding sites for different domains of p65 to TER are shown. Facilitation of TERT binding to p65-TER complex leading to hierarchical catalytic core assembly is shown. The conserved secondary structural elements of TER are marked in Figure 1B.