Post-transcriptional controls in bacteriophage T4: roles of the sequence-specific endoribonuclease RegB

Abstract: Gene regB of bacteriophage T4 encodes a sequence-specific endoribonuclease which introduces cuts in early phage messenger RNAs. In most cases, cutting takes place in the middle of the tetranucleotide GGAG. Efficient cleavages occur in the motifs located in intergenic regions, some of them being Shine-Dalgarno sequences. When located in a coding sequence, this tetranucleotide is poorly recognized or not at all. In this article, we have reviewed the properties of the RegB endoribonuclease, with emphasis on its possible roles in T4 development. We show that the nuclease RegB plays at least two roles: (i) it inactivates a sub-class of early mRNA by cleaving Shine-Dalgarno sequences, and (ii) it is necessary for the degradationn of early mRNAs, but not of middle and late mRNAs. Accordingly, we found that middle and late mRNAs avoid processing by RegB, probably for different reasons. Most of the middle mRNAs (mRNAs initiated at MotA-dependent promoters) do not contain the motif GGAG in their intergenic regions, whereas about one-third of the late genes have this motif as Shine-Dalgarno sequence. It is not yet known whether the RNase is inactivated early in the phage cycle, or whether it remains active but cannot recognize late mRNAs as substrates.     

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.     

Activation of RegB endoribonuclease by S1 ribosomal protein requires an 11 nt conserved sequence

The T4 RegB endoribonuclease cleaves specifically in the middle of the -GGAG- sequence, leading to inactivation and degradation of early phage mRNAs. In vitro, RegB activity is very weak but can be enhanced 10- to 100-fold by the Escherichia coli ribosomal protein S1. Not all RNAs carrying the GGAG motif are cleaved by RegB, suggesting that additional information is required to obtain a complete RegB target site. In this work, we find that in the presence of S1, the RegB target site is an 11 nt long single-stranded RNA carrying the 100% conserved GGA triplet at the 5′ end and a degenerate, A-rich, consensus sequence immediately downstream. Our data support the notion that RegB alone recognizes only the trinucleotide GGA, which it cleaves very inefficiently, and that stimulation of RegB activity by S1 depends on the nucleotide immediately 3′ to -GGA-.     

First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra

The bacteriophage T4 genome-encoded ribonuclease RegB is the unique well-defined restriction endoribonuclease. This protein cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence (required for the prokaryotic initiation of the translation). This protein has no significant homology to any known ribonuclease and its structure has never been investigated. The extreme toxicity of this ribonuclease prevents the expression of large quantities for structural studies. Here, we show that the toxicity of RegB can be bypassed by using the RegB H48A point mutant and explain why resolving the structure of this mutant is relevant. For nuclear magnetic resonance (NMR) purposes, we report the preparation of highly pure (13)C/(15)N double-labelled 1.2mM samples of RegB H48A using a high yield expression procedure in minimal medium (30 mg/L). We also present a set of solution conditions that maintain the concentrated samples of this protein stable for long periods at the NMR-required temperature. Finally, we present the first (1)H/(15)N and (1)H/(13)C two-dimensional NMR spectra of RegB H48A. These spectra show that the protein is folded and that the full structural analysis of RegB by NMR is feasible.     

The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using ³¹P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2′,3′-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by ¹H/¹⁵N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.     

Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependant manner

The RegB protein, encoded by the T4 bacteriophage genome, is a ribonuclease involved in the inactivation of the phage early messenger RNAs. Its in vitro activity is very low but can be enhanced up to 100-fold in the presence of the ribosomal protein S1. The latter is made of six repeats of a conserved module found in many other proteins of RNA metabolism. Considering the difference between its size (556 amino acids) and that of several RegB substrates (10 nucleotides), we wondered whether all six modules are necessary for RegB activation. We studied the influence of twelve S1 fragments on the cleavage efficiency of three short substrates. RegB activation requires the cooperation of different sets of modules depending on the substrates. Two RNAs are quite well cleaved in the presence of the fragment formed by the fourth and fifth modules, whereas the third requires the presence of the four C-terminal domains. However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or tetra-modules are similar, suggesting a common interaction surface. In the case of the tetra-module the interactions involve all four domains, raising the question of the spatial organization of this region.     

Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop

Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA. Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AGNN. Using a synthetic RNA modeled after the 25S rRNA 3' ETS cleavage site we find that the AGNN loop is an important determinant of substrate selectivity. When this loop sequence is altered, the rate of Rnt1p cleavage is reduced. The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay. Deletion of the nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage. Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay. We propose that a major determinant of AGNN loop recognition by Rnt1p is present in its dsRBD.     

The Lin28 cold-shock domain remodels pre-let-7 microRNA

The RNA-binding protein Lin28 regulates the processing of a developmentally important group of microRNAs, the let-7 family. Lin28 blocks the biogenesis of let-7 in embryonic stem cells and thereby prevents differentiation. It was shown that both RNA-binding domains (RBDs) of this protein, the cold-shock domain (CSD) and the zinc-knuckle domain (ZKD) are indispensable for pri- or pre-let-7 binding and blocking its maturation. Here, we systematically examined the nucleic acid-binding preferences of the Lin28 RBDs and determined the crystal structure of the Lin28 CSD in the absence and presence of nucleic acids. Both RNA-binding domains bind to single-stranded nucleic acids with the ZKD mediating specific binding to a conserved GGAG motif and the CSD showing only limited sequence specificity. However, only the isolated Lin28 CSD, but not the ZKD, can bind with a reasonable affinity to pre-let-7 and thus is able to remodel the terminal loop of pre-let-7 including the Dicer cleavage site. Further mutagenesis studies reveal that the Lin28 CSD induces a conformational change in the terminal loop of pre-let-7 and thereby facilitates a subsequent specific binding of the Lin28 ZKD to the conserved GGAG motif.     

Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria utilizes a type III secretion (T3S) system to inject effector proteins into eukaryotic cells. T3S substrate specificity is controlled by HpaC, which promotes secretion of translocon and effector proteins but prevents efficient secretion of the early substrate HrpB2. HpaC and HrpB2 interact with the C-terminal domain (HrcU(C) ) of the FlhB/YscU homologue HrcU. Here, we provide experimental evidence that HrcU is proteolytically cleaved at the conserved NPTH motif, which is required for binding of both HpaC and HrpB2 to HrcU(C) . The results of mutant studies showed that cleavage of HrcU contributes to pathogenicity and secretion of late substrates but is dispensable for secretion of HrpB2, which is presumably secreted prior to HrcU cleavage. The introduction of a point mutation (Y318D) into HrcU(C) activated secretion of late substrates in the absence of HpaC and suppressed the hpaC mutant phenotype. However, secretion of HrpB2 was unaffected by HrcU(Y318D) , suggesting that the export of early and late substrates is controlled by independent mechanisms that can be uncoupled. As HrcU(Y318D) did not interact with HrpB2 and HpaC, we propose that the substrate specificity switch leads to the release of HrcU(C) -bound HrpB2 and HpaC.     

The His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin

The amino acid sequence His-Pro-Phe as N-terminal residues 6-8 of the natural renin substrate, angiotensinogen, is conserved among species. We investigated whether this His-Pro-Phe motif functions as the determinant of the substrate specificity of renin. Mutant angiotensinogens in which the Ile-His-Pro-Phe-His-Leu sequence at positions 5-10 of wild-type angiotensinogen was replaced by either His-Pro-Phe-His-Leu-Leu or Ala-Ile-His-Pro-Phe-His were cleaved by renin at the C-terminal side of residues 9 and 11, respectively, while wild-type angiotensinogen was cleaved at residue 10. A triple Ala substitution for the His-Pro-Phe motif of angiotensinogen prevented its cleavage by renin. In contrast, triple Ala substitution for residues 9-11, including the natural site of cleavage by renin, allowed cleavage between the two Ala residues at positions 10 and 11. Furthermore, the 33-residue C-terminal peptide of human megsin, which carries a naturally occurring His-Pro-Phe sequence, was cleaved by renin at the C-terminal side of the His-Pro-Phe-Leu-Phe sequence. These results indicate that the His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin. By binding to a corresponding pocket on renin, the His-Pro-Phe motif may act as a molecular anchor to recruit the scissile peptide bond to a favorable site for catalysis.     

Metabolic dependence of the fluidity of intact erythrocyte membrane

An S1-nuclease sensitive site exists within supercoiled plasmids containing the 5'-flanking sequences of the human beta-globin gene. This site is located approximately 540 base pairs upstream from the start of the gene within a region of 52 alternating purine-pyrimidine residues which has the potential to adopt either cruciform structures or Z-form DNA. This site is protected from specific cleavage by S1-nuclease by the high-mobility-group chromosomal proteins HMG1 and 2, which may be specifically acting to protect short sequences of single-stranded DNA.     

Involvement of a conserved GFG motif region in substrate binding by RseP,an Escherichia coli S2P protease

RseP, an Escherichia coli S2P family intramembrane cleaving protease, is involved in regulation of the extracytoplasmic stress response and membrane quality control through specific cleavage of substrates. Recent research suggested that the PDZ domains and the MRE β‐loop (m embrane‐r ee ntrant β‐loop) are involved in substrate discrimination; the former would serve to prevent cleavage of substrates with a large periplasmic domain, whereas the latter would directly interact with the substrate's transmembrane segment and induce its conformational change. However, the mechanisms underlying specific substrate recognition and cleavage by RseP are not fully understood. Here, the roles of the N‐terminal part of the first cytoplasmic loop region (C1N) of RseP that contains a highly conserved GFG motif were investigated. A Cys modifiability assay suggested that C1N is partly membrane‐inserted like the MRE β‐loop. Pro, but not Cys, substitutions in the GFG motif region compromised the proteolytic function of RseP, suggesting the importance of a higher order structure of this motif region. Several lines of evidence indicated that the GFG motif region directly interacts with the substrate and also aids the function of the MRE β‐loop that participates in substrate recognition by RseP. These findings provide insights into the substrate recognition mechanisms of S2P proteases.     

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.     

Characterizing the function and structural organization of the 5' tRNA-like motif within the hepatitis C virus quasispecies

Hepatitis C virus (HCV) RNA is recognized and cleaved in vitro by RNase P enzyme near the AUG start codon. Because RNase P identifies transfer RNA (tRNA) precursors, it has been proposed that HCV RNA adopts structural similarities to tRNA. Here, we present experimental evidence of RNase P sensitivity conservation in natural RNA variant sequences, including a mutant sequence (A368–G) selected in vitro because it presented changes in the RNA structure of the relevant motif. The variation did not abrogate the original RNase P cleavage, but instead, it allowed a second cleavage at least 10 times more efficient, 4 nt downstream from the original one. The minimal RNA fragment that confers sensitivity to human RNase P enzyme was located between positions 299 and 408 (110 nt). Therefore, most of the tRNA-like domain resides within the viral internal ribosome entry site (IRES) element. In the variant, in which the mutation stabilizes a 4 nt stem–loop, the second cleavage required a shorter (60 nt) substrate, internal to the minimal fragment substrate, conforming a second tRNA-like structure with similarities to a ‘Russian-doll’ toy. This new structure did not impair IRES activity, albeit slightly reduced the efficiency of translation both in vitro and in transfected cells. Conservation of the original tRNA-like conformation together with preservation of IRES activity points to an essential role for this motif. This conservation is compatible with the presence of RNA structures with different complexity around the AUG start codon within a single viral population (quasispecies).     

Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme

The 5' capping of mammalian pre-mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 A crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5' triphosphate end. Structural, biochemical and mutational results show that despite sharing an HCxxxxxR(S/T) motif, a phosphoenzyme intermediate and a core alpha/beta-fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by mammalian capping enzyme differs from that used by protein phosphatases to hydrolyze phosphomonoesters. The most significant difference is the absence of a carboxylate general acid catalyst in RNA triphosphatase. Residues conserved uniquely among the RNA phosphatase subfamily are important for function in cap formation and are likely to play a role in substrate recognition.     

Mapping of the major early endonuclease cleavage site of the rat precursor to rRNA within the internal transcribed spacer sequence of rDNA

The endonuclease cleavage of 41 S pre-rRNA to yield 32 S and 21 S pre-rRNA constitutes a major early step in the processing of pre-rRNA in rat liver. The 5'-terminus of 32 S pre-rRNA and the 3'-terminus of 21 S pre-rRNA were precisely located within the rDNA sequence by S1 nuclease protection mapping and use of appropriate rDNA restriction fragments. The 5'-terminus of 12 S pre-rRNA, an initial product of 32 S pre-rRNA processing, was also mapped within the rDNA sequence. The 5'-termini of 32 S and 12 S pre-rRNA coincide and map within a 14-residue T-tract (non-coding strand) at 161-163 bp upstream from the 5'-end of the 5.8 S rRNA gene. The 3'-terminus of 21 S pre-rRNA maps within the same T-tract. These results show that the endonuclease cleavage occurs within a U-tract in the internal transcribed spacer 1 sequence of 41 S pre-rRNA. The homogeneity of the 5'- or 3'-termini of 32 S, 12 S and 21 S pre-rRNA indicates also that the terminal processing of these molecules, if any, is markedly slower. The coincidence in the location of 32 S and 12 S pre-rRNA 5'-termini shows further that the endonuclease cleavage of 32 S pre-rRNA precedes the removal of its 5'-terminal segment to yield 5.8 S rRNA. The absence in the whole pre-rRNA internal transcribed spacer of sequences complementary to the target U-tract suggests that the endonuclease cleavage, generating 32 S and 21 S pre-rRNA, occurs in a single-stranded loop of U-residues.