Lsm proteins and Hfq: Life at the 3' end

Abstract

The bacterial Hfq protein is a versatile modulator of RNA function and is particularly important for regulation mediated by small non-coding RNAs. Hfq is a bacterial Sm protein but bears more similarity to the eukaryotic Sm-like (Lsm) family of proteins than the prototypical Sm proteins. Hfq and Lsm proteins share the ability to chaperone RNA-RNA and RNA/protein interactions and an interesting penchant for protecting the 3' end of a transcript from exonucleolytic decay while encouraging degradation through other pathways. Our view of Lsm function in eukaryotes has historically been informed by studies of Hfq structure and function but mutational analyses and structural studies of Lsm sub-complexes have given important insights as well. Here, we aim to compare and contrast the roles of these evolutionarily related complexes and to highlight areas for future investigation.

Keywords: RNA chaperone; Sm-like; exoribonuclease; mRNA decay; oligouridylation; polyadenylation; splicing.

Figure 1. Schematic showing the structure of the Sm fold. The five anti-parallel β-strands and single α-helix are depicted in different colors. Loops 3 and 5 contain residues important for RNA-binding. Loop 4 varies in length and amino acid composition.

Figure 2. Hfq and Lsm proteins bind single-stranded 3′ ends and activate RNA decay. (A) Poly(A) polymerase (PAP) acts distributively to add adenosine residues until the poly(A) tail reaches sufficient length to recruit Hfq. Hfq binds poly(A) through its distal surface and stimulates PAP activity to extend the poly(A) tail. This interaction also prevents the 3′-5′ exonuclease, polynucleotide phosphorylase (PNPase), from attacking the 3′ end of the transcript.^- (B) In eukaryotic cells, poly(A) tails are shortened by deadenylase enzymes prior to decay. Once the tail is short enough (~10–12 residues), Lsm1-7 associates using the proximal binding surface and Pat1 is also recruited. The Lsm-Pat1 complex prevents further shortening from the 3′ end while activating decapping at the 5′ end by interacting with a number of accessory factors to engage the decapping enzyme (Dcp2). (C) Bacterial sRNAs terminate in a tract of seven to nine uridines generated through Rho-independent termination. This 3′ U-tract is recognized by Hfq through its proximal binding surface resulting in protection of the 3′OH group from attack by PNPase. Additional regions of the sRNA can bind the lateral surface of Hfq while the distal surface recognizes mRNA targets. Once the mRNA and sRNA anneal, RNase E joins the complex and cleaves both the mRNA and sRNA to initiate their degradation.^,, (D) Vertebrate histone mRNAs are degraded through a pathway that requires 3′ oligouridylation by a terminal uridyltransferase (TUTase).^, This provides a platform to recruit the Lsm1–7/Pat1 complex which, in turn, initiates mRNA decapping.^, Oligouridylation of histone mRNAs also promotes 3′-5′ exonucleolytic decay as Lsm1-7 recruits the Eri1 exonuclease.

Figure 3. U6 snRNP metabolism requires Lsm2-8. U6 snRNA is transcribed in the nucleus by RNA polymerase III and terminates in four uridine residues, which recruit the La RNA-binding protein. The U tract is extended by U6 TUTase leading to exchange of La for Lsm2-8.^, The U-tract is then trimmed to five residues by Mpn1 and consequently terminates in a 2’,3′ cyclic phosphate.^, Lsm binding allows association of Prp24/p110 to generate mature U6 snRNA. Lsm facilitates annealing of U4 and U6 and the complex enters the spliceosome. Subsequent rearrangements allow U6 to interact with U2 and the pre-mRNA and Lsm2-8 and p110 are then ejected prior to the first step of splicing. After splicing, U6 is released in a form that is accessible to various RNA-modifying enzymes. U6 snRNP must then be reassembled prior to being re-used. Lsm2-8 and the U6 TUTase may participate in a quality control mechanism to ensure the U-tract remains intact.

Figure 4. Conservation of RNA-binding residues in Lsm and Hfq proteins. (A) Alignment of primary sequence of Sm domains for human Lsm1-8 proteins, S. cerevisiae Lsm1 and Hfq from E. coli and S. aureus. C-terminal domains and some N-terminal sequences are excluded from this alignment for clarity. The alignment was generated using CLUSTAL-W and edited in JalView. Secondary structure is indicated below the alignment and colored as in Figure 1. Residues shown to be important for RNA-binding are highlighted (Hfq) or indicated by arrows (Lsm proteins). Conserved residues are indicated by blue shading with darker blue denoting more conservation. The accession numbers for the protein sequences used in the alignment are: H.s. Lsm1 NP_055277.1, S.c. Lsm1 NP_012411.1, H.s. Lsm2 NP_067000.1, H.s. Lsm3 NP_055278.1, H.s. Lsm4 NP_036453.1, H.s. Lsm5 NP_036454.1, H.s. Lsm6 NP_009011.1, H.s. Lsm7 NP_057283.1, H.s. Lsm8 NP_057284.1, E.c.Hfq ACE63256.1, S.a.Hfq AEW65270.1. (B) Space-filling structure of proximal surface of S. Hfq (PDB ID:2YLB). Residues involved in RNA-binding are highlighted in yellow. (C) Space-filling structure of proximal surface of an S. cerevisiae Lsm3 octamer (PDB ID: 3BW1). Residues likely to be involved in RNA recognition are highlighted in yellow. The images in (B and C) were generated using Cn3D Structure Viewer Ver 4.3.