The N-terminal domains of FLASH and Lsm11 form a 2:1 heterotrimer for histone pre-mRNA 3'-end processing

Abstract

Unlike canonical pre-mRNAs, animal replication-dependent histone pre-mRNAs lack introns and are processed at the 3'-end by a mechanism distinct from cleavage and polyadenylation. They have a 3' stem loop and histone downstream element (HDE) that are recognized by stem-loop binding protein (SLBP) and U7 snRNP, respectively. The N-terminal domain (NTD) of Lsm11, a component of U7 snRNP, interacts with FLASH NTD and these two proteins recruit the histone cleavage complex containing the CPSF-73 endonuclease for the cleavage reaction. Here, we determined crystal structures of FLASH NTD and found that it forms a coiled-coil dimer. Using solution light scattering, we characterized the stoichiometry of the FLASH NTD-Lsm11 NTD complex and found that it is a 2:1 heterotrimer, which is supported by observations from analytical ultracentrifugation and crosslinking.

Fig 1. Multiple protein sequence alignment of FLASH and Lsm11 N-terminal domains.

(A) Lsm11 N-terminal domain. (B) FLASH N-terminal domain. Alignment was carried out with Clustal Omega [60, 61] and the results displayed with ESPript [62]. Secondary structure for FLASH is based on the structure of human FLASH NTD, while that for Lsm11 is based on Psipred [63] secondary structure prediction of human Lsm11. Conserved residues are highlighted in red with white fonts, semi-conserved residues in red fonts, and other residues in black fonts. Blue dots indicate residues at the FLASH dimer interface. Gaps are indicated by dotted lines. Species abbreviations: Hs, Homo sapiens (human); Dr, Danio rerio (zebrafish); Dm, Drosophila melanogaster (fruit fly).

Fig 2. Structure of FLASH NTD dimer.

(A) Two views of the FLASH NTD forming a coiled-coil dimer with respective protomers colored in green and yellow orange. The hexahistidine-tag was observed in protomer 1. Side chains of residues involved in the dimer interface are shown as sticks, while the other side chains are shown as thin sticks. Colors of atoms: red, oxygen; blue, nitrogen; yellow, sulfur/selenium. (B) Hydrophilic interactions (black dashes) formed by Gln100/Asn101 and Glu107/Asn108 respectively. (C) 2F_o–F_c electron density for the Cys83 side chains, contoured at 1σ (blue).

Fig 3. Structures of FLASH NTD C54S/C83A dimer.

(A) Structure of FLASH NTD C54S/C83A crystal form 1 (resolution 2.1 Å) showing FLASH dimer without the presence of a disulfide bond. (B) Structure of FLASH NTD C54S/C83A crystal form 2 (resolution: 2.6 Å) showing observable residues 52–70 that adopt a helical structure on protomer 1, and are less ordered in protomer 2. The LDLY motif essential for binding the HCC is shown as sticks. (C) Superimposition of the structures of FLASH NTD C54S/C83A crystal forms 1 (cyan) and 2 (gray) with wild-type FLASH NTD dimers (green).

Fig 4. Biophysical analysis of FLASH NTD mutants.

(A) Close-up view of Leu118 and Ile119 with 2F_o–F_c electron density of the side chains (sticks) contoured at 1σ (blue). Coordinates and the electron density are derived from structure of FLASH NTD C54S/C83A crystal form 1 at 2.1 Å resolution. (B) Superose-12 analytical gel filtration profiles of FLASH NTD proteins. Peak heights are scaled to an arbitrary unit of 100. (C) SDS-PAGE analysis of co-purification of His-tagged Lsm11 NTD and wild-type and mutant FLASH NTD. All FLASH mutants have additional C54S/C83A mutations. Pellet and supernatant are the insoluble and soluble fractions, respectively, of the cell lysate.

Fig 5. Size exclusion chromatograph-multi-angle light scattering of FLASH NTD-Lsm11 NTD complex and FLASH NTD C54S/C83A.

SEC-MALS traces (with superimposed calculated molar mass traces) of FLASH NTD-Lsm11 NTD complex at 500 mM NaCl (blue), FLASH NTD C54S/C83A at 500 mM NaCl (purple), FLASH NTD-Lsm11 NTD complex at 250 mM NaCl (red), and FLASH NTD C54S/C83A at 250 mM NaCl (green). Left axis, molar mass; right axis, light scattering signal; bottom axis, elution volume.

Fig 6. Analytical ultracentrifugation studies of FLASH NTD, FLASH NTD mutants, Lsm11, and FLASH NTD-Lsm11 NTD complex.

(A) Typical traces of absorbance at 280 nm of the protein in 20 mM Tris (pH 7.5) buffer during the sedimentation velocity experiment. The protein concentration was 1 mg/ml. For clarity, only every fifth scan is shown. The symbols represent experimental data and the lines are the results obtained after being fitted to the Lamm equation using the SEDFIT program [57]. (B-E) Continuous c(s) distribution of FLASH NTD wild-type, C54S/C83A mutant, FLASH NTD-Lsm11 NTD complex and Lsm11 NTD. The distributions of the proteins at concentrations of 1 mg/ml (B-D) and 0.8 mg/ml (E) are shown by solid lines and those at concentrations of 0.1 mg/ml (B) and 0.05 mg/ml (D) are shown by dashed lines and that at 8 mg/ml (D) are showed by dotted line. The y-axis on the right is for the protein at a concentration of 0.1 mg/ml (B) and 8 mg/ml (D). The vertical dashed lines on the left and right indicate the monomer position of Lsm11 NTD and the dimer position of FLASH NTD, respectively. The residual bitmap of the raw data and the best-fit results are shown in the insets. The data are summarized in Table 2.