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.

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

“…The analysis of the 3D structure indicates that each fragment folds into three α-helices, resembling the Myb DNA binding domain and SANT domain [23], consistent with previously published structural reports [30]. In agreement with the significant sequence homology (45% of identical amino acids) ( Figure S4A), the structures of the FLASH and YARP fragments are very similar with r.m.s.d.…”

“…The U7 snRNP consists of an approximately 60-nucleotide U7 snRNA and a heptameric Sm ring characterized by the presence of two unique proteins: Lsm10 and Lsm11 [19,20]. Lsm11 through its extended N-terminal region interacts with the N-terminal region of FLASH, a protein of 220 kDa, and together they recruit a complex of multiple polyadenylation factors to the U7 snRNP [21][22][23]. One of these factors, CPSF73, functions as the processing endonuclease that cleaves histone pre-mRNA at the 3' end [24].…”

Studies with recombinant U7 snRNP demonstrate that CPSF73 is both an endonuclease and a 5′–3′ exonuclease

“…Subunits of the U7-specific Sm ring were expressed either in bacteria (SmB,SmD3,SmE,SmF,and SmG) or in insect cells using the baculovirus system (Lsm10 and Lsm11 with an internal deletion of amino acids 211-332), and assembled on U7 snRNA yielding core U7 snRNP, as described (Bucholc et al 2020). The core U7 snRNP was either directly purified by size exclusion column (SEC), or prebound to bacterially expressed FLASH encompassing amino acids 51-137 (Aik et al 2017;Sun et al 2020), and the resultant complex was stored at −80°C, as described (Bucholc et al 2020;Sun et al 2020). SLBP (amino acids 125-270) with an amino-terminal 6xHis tag was expressed in bacteria (Bucholc et al 2020).…”

“…Lsm10 and Lsm11 are unique to U7 snRNP, replacing the SmD1 and SmD2 subunits found at the same positions in the spliceosomal snRNPs (Pillai et al 2001(Pillai et al , 2003. Lsm11 through its long amino-terminal region interacts with the amino-terminal region of the 200 kDa protein FLASH (Yang et al 2009a;Aik et al 2017) and the two proteins form a platform that recruits the Histone pre-mRNA Cleavage Complex (HCC), a subset of proteins shared with the canonical cleavage and polyadenylation machinery (Yang et al 2009a(Yang et al , 2013. Analysis of purified Drosophila and mouse U7 snRNPs by mass spectrometry identified CPSF73, CPSF100, symplekin and CstF64 as the major components of the HCC, with the remaining CPSF subunits (CPSF160, WDR33, Fip1, and CPSF30) being present in much smaller amounts (Sabath et al 2013;Skrajna et al 2018).…”

Structure of an active human histone pre-mRNA 3′-end processing machinery

“…To prepare a fully recombinant machinery, we reconstituted human U7 snRNP (13) and mixed it with purified human HCC, FLASH (14), and SLBP (15). Using a modified mouse histone H2a pre-mRNA (H2a*) (fig.…”