Structure of S. pombe telomerase protein Pof8 C-terminal domain is an xRRM conserved among LARP7 proteins

Abstract

La-related proteins 7 (LARP7) are a class of RNA chaperones that bind the 3' ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In the ciliate Tetrahymena the LARP7 protein p65 is a component of telomerase, an essential ribonucleoprotein complex that maintains the telomeric DNA at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase. Unexpectedly, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. LARP7 proteins have a conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and human Larp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix α3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.

Keywords: 7SK; LARP; La protein; NMR; RNA; RRM; X-ray crystallography; telomerase; xRRM.

Figure 1.

Domains and sequence alignments of LARP7 and La proteins (A) Domain organization of LARP7s from yeast S. pombe (Pof8), ciliate Euplotes aediculatus (p43), ciliate Tetrahymena thermophila (p65), and human (Larp7). Numbers above the sequence schematic indicate known domain boundaries of the La motif (cyan), RRM1 (green) and xRRM (orange). (B) Sequence alignment of LARP7 p65 and Larp7 xRRMs, human La protein (hLa) RRM2, Pof8, and Euplotes p43. Residues with high similarity are coloured red. The secondary structure elements of Pof8 and hLarp7 are shown as helices and sheets above and below the sequence, respectively. Locations of RNP1 and RNP2 in canonical RRMs are indicated above the sequence in brackets for reference. Conserved residues determined by this and previous work to contribute to RNA binding and helix α3–β sheet interaction are sky blue and orange (for β-sheet) and red (for α3), respectively. (C) Cartoon comparing the conserved RNA binding features of a canonical RRM (left) and xRRM (right), based in part on this work. Conserved residues that bind RNA are shown in sky blue and that contribute to the helix α3–β sheet interaction are shown in red and orange, respectively. For the canonical RRM, circled residues are those that most commonly interact with RNA. For the xRRM, the length of helix α3 (shown in grey) varies. Variable regions not involved in RNA binding (β4′ strand and loops) not pictured

Figure 2.

Crystal structure of Pof8 RRM2 at 1.35 Å. (A) 2 Fo-Fc electron density map with crystal structure model shown in stick representation. The map is contoured at 2σ. (B, C) Two views of ribbon representation of the Pof8 xRRM (residues 288–402) crystal structure. The β-sheet is coloured orange, helices α1 and α2 are tan, and helix α3 is red. (D) Ribbon representation with equivalent conserved residues involved in RNA binding in p65 and hLarp7 xRRMs shown as sticks. (E) Ribbon representation with conserved residues involved in stabilizing the α3–β-sheet interactions shown as sticks. (F) Ribbon representation with conserved residues involved in stabilizing the α3–β-sheet interactions shown as space fill to highlight stacking interactions

Figure 3.

NMR characterization of Pof8 RRM2 (A) ¹H-¹⁵N HSQC spectrum of Pof8 xRRM. Pairs of amide peaks that are doubled are circled; those for which the minor peak is assigned are labelled in red and inferred by proximity to major peak in orange. (B) Expanded regions from panel A showing peak doubling of amides W397 (helix α3) and I341 (β3). (C) Distribution of residues whose amides show peak doubling, mapped onto the structure. Assigned residues are red, residues with inferred assignments are orange. (D) Plot of heteronuclear NOE values vs residue number. Secondary structure elements are indicated above. (E) Plot of normalized order parameters vs residue number. Residues with values greater than 1.5 are labelled inset. (F) ¹H-¹⁵N heteronuclear NOE values mapped onto a ribbon structure, with scale ranging from grey (na, not available), 0.5 (green) to 1.0 (blue). (G) Crystal structure B-factor mapped on a ribbon structure, scale ranging from 15.00 (blue) to 70.00 (red)

Figure 4.

Comparison of structures of LARP7 xRRMs and hLa protein RRM2: helix α3–β sheet interactions. Ribbon representations of RRM2 from (A) S. pombe Pof8, crystal structure (this work), (B) Tetrahymena p65, crystal structure (PDB ID 4EYT) and (C) human Larp7, solution NMR structure (PDB ID 5KNW), (D) human La, solution NMR structure (PDB ID 1OWX). Dashed line indicates disordered residues missing in the density and/or that become helical on binding RNA. (E-H) Zoomed regions of (A-D) highlighting the conserved residues important for α3–β-sheet interactions. The first three turns of helix α3 are numbered. Secondary structure motifs are coloured as in Fig 2. Residue side chains important for α3–β sheet interactions are shown as ball and stick and coloured green (hydrophobic) and cyan (charged)

Figure 5.

Comparison of structures of LARP7 xRRMs: RNA binding determinants (A-C) Ribbon representations of crystal structures of (A) Pof8 xRRM (this work), (B) p65 xRRM bound to telomerase RNA stem 4 (PDB ID 4ERD), (C) hLarp7 xRRM bound to 7SK stem-loop 4 (PDB ID 6D12). Side chains that interact (B, C) or are predicted to interact (A) with RNA are shown as sticks; (D-F) Surface representations of (D) Pof8 xRRM (this work), (E) p65 xRRM bound to telomerase RNA stem 4 (PDB ID 4ERD), (F) hLarp7 xRRM bound to 7SK stem-loop 4 (PDB ID 6D12), rotated 90° from (A-C). The proposed RNA interacting surface of Pof8 is shown by the grey arc, and for p65 and hLarp7 the interacting RNA nucleotides are shown as sticks, with Gua in green, and an RNA schematic is shown at right; (G) Stick representation of Gua recognition in the xRRM binding pocket. The interaction between β3 R and a Ura O4 (e.g. hLarp7) or Ade N7 (e.g. p65) is also indicated; (H) Ribbon representation of Pof8 with residues whose substitution to alanine or deletion affect RNA binding in vitro or TER abundance and telomere length in vivo [15–17] shown as sticks. Δα3 is deletion of the entire helix. I341A/I342A is a double substitution. (I-J) Ribbon and stick representation illustrating position of RNP3 Y on (I) RNA free and bound p65 xRRM (gold) and RNA free Pof8 xRRM (orange) and (J) RNA free and bound hLarp7 xRRM (argon blue) and RNA free Pof8 xRRM (orange)