Crystal structure of the PIN domain of human telomerase-associated protein EST1A

Saccharomyces cerevisiae Est1p is a telomerase-associated protein essential for telomere length homeostasis. hEST1A is one of the three human Est1p homologues and is considered to be involved not only in regulation of telomere elongation or capping but also in nonsense-mediated degradation of RNA. hEST1A is composed of two conserved regions, Est1p homology and PIN (PilT N-terminus) domains. The present study shows the crystal structure of the PIN domain at 1.8 A resolution. The overall structure is composed of an alpha/beta fold or a core structure similar to the counterpart of 5' nucleases and an extended structure absent from archaeal PIN-domain proteins and 5' nucleases. The structural properties of the PIN domain indicate its putative active center consisting of invariant acidic amino acid residues, which is geometrically similar to the active center of 5' nucleases and an archaeal PAE2754 PIN-domain protein associated with exonuclease activity.     

Structural conservation of the PIN domain active site across all domains of life

The PIN (PilT N‐terminus) domain is a compact RNA‐binding protein domain present in all domains of life. This 120‐residue domain consists of a central and parallel β sheet surrounded by α helices, which together organize 4–5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity. In bacteria and archaea, the PIN domain is primarily associated with toxin–antitoxin loci, consisting of a toxin (the PIN domain nuclease) and an antitoxin that inhibits the function of the toxin under normal growth conditions. During nutritional or antibiotic stress, the antitoxin is proteolytically degraded causing activation of the PIN domain toxin leading to a dramatic reprogramming of cellular metabolism to cope with the new situation. In eukaryotes, PIN domains are commonly found as parts of larger proteins and are involved in a range of processes involving RNA cleavage, including ribosomal RNA biogenesis and nonsense‐mediated mRNA decay. In this review, we provide a comprehensive overview of the structural characteristics of the PIN domain and compare PIN domains from all domains of life in terms of structure, active site architecture, and activity.     

Structures of the PIN domains of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex

SMG6 and SMG5 are essential factors in nonsense-mediated mRNA decay, a conserved pathway that degrades mRNAs with premature translation termination codons. Both SMG5 and SMG6 have been predicted to contain a C-terminal PIN (PilT N-terminus) domain, present in proteins with ribonuclease activity. We have determined the structures of human SMG5 and SMG6 PIN domains. Although they share a similar overall fold related to ribonucleases of the RNase H family, they have local differences at the putative active site. SMG6 has the canonical triad of acidic residues that are crucial in RNase H for nuclease activity, while SMG5 lacks key catalytic residues. The structural differences are reflected at the functional level. Only the PIN domain of SMG6 has degradation activity on single-stranded RNA in vitro. This difference in catalytic activity is conserved in Drosophila, where an SMG6 with an inactive PIN domain inhibits NMD in a dominant-negative manner. Our findings suggest that the NMD machinery has intrinsic nuclease activity that is likely to contribute to the rapid decay of mRNAs that terminate translation prematurely.     

Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing

Human Tudor-SN is involved in the degradation of hyper-edited inosine-containing microRNA precursors, thus linking the pathways of RNA interference and editing. Tudor-SN contains four tandem repeats of staphylococcal nuclease-like domains (SN1–SN4) followed by a tudor and C-terminal SN domain (SN5). Here, we showed that Tudor-SN requires tandem repeats of SN domains for its RNA binding and cleavage activity. The crystal structure of a 64-kD truncated form of human Tudor-SN further shows that the four domains, SN3, SN4, tudor and SN5, assemble into a crescent-shaped structure. A concave basic surface formed jointly by SN3 and SN4 domains is likely involved in RNA binding, where citrate ions are bound at the putative RNase active sites. Additional modeling studies provide a structural basis for Tudor-SN's preference in cleaving RNA containing multiple I·U wobble-paired sequences. Collectively, these results suggest that tandem repeats of SN domains in Tudor-SN function as a clamp to capture RNA substrates.     

How an exonuclease decides where to stop in trimming of nucleic acids: crystal structures of RNase T-product complexes

Exonucleases are key enzymes in the maintenance of genome stability, processing of immature RNA precursors and degradation of unnecessary nucleic acids. However, it remains unclear how exonucleases digest nucleic acids to generate correct end products for next-step processing. Here we show how the exonuclease RNase T stops its trimming precisely. The crystal structures of RNase T in complex with a stem-loop DNA, a GG dinucleotide and single-stranded DNA with different 3'-end sequences demonstrate why a duplex with a short 3'-overhang, a dinucleotide and a ssDNA with a 3'-end C cannot be further digested by RNase T. Several hydrophobic residues in RNase T change their conformation upon substrate binding and induce an active or inactive conformation in the active site that construct a precise machine to determine which substrate should be digested based on its sequence, length and structure. These studies thus provide mechanistic insights into how RNase T prevents over digestion of its various substrates, and the results can be extrapolated to the thousands of members of the DEDDh family of exonucleases.     

Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases.

The 3'-->5' exonucleases catalyze the excision of nucleoside monophosphates from the 3' termini of DNA. We have identified the cDNA sequences encoding two 3'-->5' exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 3'-->5' exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3' terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.     

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.     

Functional analysis of the domain organization of Trypanosoma brucei RNase HI

The structure-function relationship of Trypanosoma brucei RNase HI was investigated by evaluating the abilities of truncated forms of the enzyme to convert RNase H substrate to product. Our studies identify a 42-amino-acid noncanonical RNase HI spacer domain essential for function. We also show that the enzyme's nuclear localization domain is not required for RNase H activity but functions as an RNA binding domain which modulates the enzyme's Mn(2+)-dependent activity. These findings show that the enzyme's RNA binding/nuclear targeting and RNase H activities are organized into discrete N- and C-terminal domains with boundaries established by its spacer domain. This is the first report of the unusual structure to function relationship of a protozoal RNase H. This relationship may be conserved in other eukaryotic RNases H suggesting that criteria preserving their structure and function may be important to their roles in nucleic acid metabolism.     

Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 Å resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3′ overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 Å, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.     

Structural basis for RNA trimming by RNase T in stable RNA 3'-end maturation

RNA maturation relies on various exonucleases to remove nucleotides successively from the 5' or 3' end of nucleic acids. However, little is known regarding the molecular basis for substrate and cleavage preference of exonucleases. Our biochemical and structural analyses on RNase T-DNA complexes show that the RNase T dimer has an ideal architecture for binding a duplex with a short 3' overhang to produce a digestion product of a duplex with a 2-nucleotide (nt) or 1-nt 3' overhang, depending on the composition of the last base pair in the duplex. A 'C-filter' in RNase T screens out the nucleic acids with 3'-terminal cytosines for hydrolysis by inducing a disruptive conformational change at the active site. Our results reveal the general principles and the working mechanism for the final trimming step made by RNase T in the maturation of stable RNA and pave the way for the understanding of other DEDD family exonucleases.     

Resonance assignments of a putative PilT N-terminus domain protein SSO1118 from hyperthermophilic archaeon Sulfolobus solfataricus P2

PilT N-terminus (PIN) domains exist broadly in all three kingdoms of life, but the functions are not clear for most of them. Archaea species often encode multiple PIN domain-containing proteins, and the signaling and stress response roles have been proposed for these proteins. Some PIN domain proteins possess nuclease activities, which were proposed to be important in toxin-antitoxin stress response, nonsense-mediated mRNA decay, or RNA interference. SSO1118 from hyperthermophilic archaeon Sulfolobus solfataricus P2 is a putative PIN domain protein with low homology to other known PIN domain proteins. Here we report the NMR resonance assignments of SSO1118 for further structural determination and functional studies. The secondary structures predicted from the assigned chemical shifts consist with those of archaeal PIN domain proteins.     

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain

Ribonuclease (RNase) P is a site‐specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix‐loop‐helix protein‐binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 Å. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.     

The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.     

Ribonuclease III: new sense from nuisance.

RNases play an important role in the processing of precursor RNAs, creating the mature, functional RNAs. The ribonuclease III family currently is one of the most interesting families of endoribonucleases. Surprisingly, RNase III is involved in the maturation of almost every class of prokaryotic and eukaryotic RNA. We present an overview of the various substrates and their processing. RNase III contains one of the most prominent protein domains used in RNA-protein recognition, the double-stranded RNA binding domain (dsRBD). Progress in the understanding of this domain is summarized. Furthermore, RNase III only recently emerged as a key player in the new exciting biological field of RNA silencing, or RNA interference. The eukaryotic RNase III homologues which are likely involved in this process are compared with the other members of the RNase III family.     

Crystal structure of Escherichia coli RNase D, an exoribonuclease involved in structured RNA processing

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.     

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of alpha-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.     

Comparative analysis of editosome proteins in trypanosomatids

Detailed comparisons of 16 editosome proteins from Trypanosoma brucei, Trypanosoma cruzi and Leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in RNA editing. Five related proteins with RNase III-like motifs also contain a U1-like zinc finger and either dsRBM or Pumilio motifs. These proteins may provide the endoribonuclease function in editing. Two other related proteins, at least one of which is associated with U-specific 3′ exonuclease activity, contain two putative nuclease motifs. Thus, editosomes contain a plethora of nucleases or proteins presumably derived from nucleases. Five additional related proteins, three of which have zinc fingers, each contain a motif associated with an OB fold; the TUTases have C-terminal folds reminiscent of RNA binding motifs, thus indicating the presence of numerous nucleic acid and/or protein binding domains, as do the two RNA ligases and a RNA helicase, which provide for additional catalytic steps in editing. These data indicate that trypanosomatid RNA editing is orchestrated by a variety of domains for catalysis, molecular interaction and structure. These domains are generally conserved within other protein families, but some are found in novel combinations in the editosome proteins.     

Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri

The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg²⁺‐dependent ribonuclease and has been shown to target initiator tRNA^fMet in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892–899. © 2016 Wiley Periodicals, Inc.