Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.     

Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea

Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent K(d) ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.     

Probing the structure and function of an archaeal C/D-box methylation guide sRNA

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus contains dozens of small C/D-box sRNAs that use a complementary guide sequence to target 2'-O-ribose methylation to specific locations within ribosomal and transfer RNAs. The sRNAs are approximately 50-60 nucleotides in length and contain two RNA structural kink-turn (K-turn) motifs that are required for assembly with ribosomal protein L7Ae, Nop5, and fibrillarin to form an active ribonucleoprotein (RNP) particle. The complex catalyzes guide-directed methylation to target RNAs. Earlier work in our laboratory has characterized the assembly pathway and methylation reaction using the model sR1 sRNA from Sulfolobus acidocaldarius. This sRNA contains only one antisense region situated adjacent to the D-box, and methylation is directed to position U52 in 16S rRNA. Here we have investigated through RNA mutagenesis, the relationship between the sR1 structure and methylation-guide function. We show that although full activity of the guide requires intact C/D and C'/D' K-turn motifs, each structure plays a distinct role in the methylation reaction. The C/D motif is directly implicated in the methylation function, whereas the C'/D' element appears to play an indirect structural role by facilitating the correct folding of the RNA. Our results suggest that L7Ae facilitates the folding of the K-turn motifs (chaperone function) and, in addition, is required for methylation activity in the presence of Nop5 and Fib.     

Comparative analysis of the 15.5kD box C/D snoRNP core protein in the primitive eukaryote Giardia lamblia reveals unique structural and functional features

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 ?. The Giardia 15.5kD protein exhibits the typical α-β-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.     

Molecular basis of box C/D RNA-protein interactions; cocrystal structure of archaeal L7Ae and a box C/D RNA

We have determined and refined a crystal structure of the initial assembly complex of archaeal box C/D sRNPs comprising the Archaeoglobus fulgidus (AF) L7Ae protein and a box C/D RNA. The box C/D RNA forms a classical kink-turn (K-turn) structure and the resulting protein-RNA complex serves as a distinct platform for recruitment of the fibrillarin-Nop5p complex. The cocrystal structure confirms previously proposed secondary structure of the box C/D RNA that includes a protruded U, a UU mismatch, and two sheared tandem GA base pairs. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct K-turn RNAs revealed a set of remarkably conserved principles in protein-RNA interactions. These analyses provide a structural basis for interpreting the functional roles of the box C/D sequences in directing specific assembly of box C/D sRNPs.     

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C′/D′ motifs. We show here that conserved terminal stem–loops formed by the C′/D′ motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem–loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles.     

Dynamic guide-target interactions contribute to sequential 2'-O-methylation by a unique archaeal dual guide box C/D sRNP

Assembly and guide-target interaction of an archaeal box C/D-guide sRNP was investigated under various conditions by analyzing the lead (II)-induced cleavage of the guide RNA. Guide and target RNAs derived from Haloferax volcanii pre-tRNA(Trp) were used with recombinant Methanocaldococcus jannaschii core proteins in the reactions. Core protein L7Ae binds differentially to C/D and C'/D' motifs of the guide RNA, and interchanging the two motifs relative to the termini of the guide RNA did not affect L7Ae binding or sRNA function. L7Ae binding to the guide RNA exposes its D'-guide sequence first followed by the D guide. These exposures are reduced when aNop5p and aFib proteins are added. The exposed guide sequences did not pair with the target sequences in the presence of L7Ae alone. The D-guide sequence could pair with the target in the presence of L7Ae and aNop5p, suggesting a role of aNop5p in target recruitment and rearrangement of sRNA structure. aFib binding further stabilizes this pairing. After box C/D-guided modification, target-guide pairing at the D-guide sequence is disrupted, suggesting that each round of methylation may require some conformational change or reassembly of the RNP. Asymmetric RNPs containing only one L7Ae at either of the two box motifs can be assembled, but a functional RNP requires L7Ae at the box C/D motif. This arrangement resembles the asymmetric eukaryal snoRNP. Observations of initial D-guide-target pairing and the functional requirement for L7Ae at the box C/D motif are consistent with our previous report of the sequential 2'-O-methylations of the target RNA.     

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense-box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion, this is the first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression.     

The crystal structure of the Methanocaldococcus jannaschii multifunctional L7Ae RNA-binding protein reveals an induced-fit interaction with the box C/D RNAs

Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that recognizes the K-turn motif in ribosomal, box H/ACA, and box C/D sRNAs. The crystal structure of Methanocaldococcus jannaschii L7Ae has been determined to 1.45 A, and L7Ae's amino acid composition, evolutionary conservation, functional characteristics, and structural details have been analyzed. Comparison of the L7Ae structure to those of a number of related proteins with diverse functions has revealed significant structural homology which suggests that this protein fold is an ancient RNA-binding motif. Notably, the free M. jannaschii L7Ae structure is essentially identical to that with RNA bound, suggesting that RNA binding occurs through an induced-fit interaction. Circular dichroism experiments show that box C/D and C'/D' RNA motifs undergo conformational changes when magnesium or the L7Ae protein is added, corroborating the induced-fit model for L7Ae-box C/D RNA interactions.     

In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNA(Trp)

Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNA(Trp) represents an outstanding specimen able to guide in cis, instead of in trans, two 2'-O-methylations in the pre-tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge-helix-bulge (BHB) splicing motif, while the RNA-guided methylation and pre-tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre-tRNA and tested its site-specific methylation activity. Recognition by L7Ae of hallmark K-turns at the C/D and C'/D' motifs appears as a crucial assembly step required for subsequent binding of a Nop5p-aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p-aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis-acting system to form guide RNA duplex(es) relative to classical trans- acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non-canonical stem of the K-turn, suggest that binding of Nop5p-aFib to the pre-tRNA-L7Ae complex might direct transition from a splicing-competent structure to an RNA conformer displaying the guide RNA duplexes required for site-specific methylation.     

Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5–guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.     

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops

The archaeal protein L7Ae and eukaryotic homologs such as L30e and 15.5kD comprise the best characterized family of K-turn-binding proteins. K-turns are an RNA motif comprised of a bulge flanked by canonical and noncanonical helices. They are widespread in cellular RNAs, including bacterial gene-regulatory RNAs such as the c-di-GMP-II, lysine, and SAM-I riboswitches, and the T-box. The existence in bacteria of K-turn-binding proteins of the L7Ae family has not been proven, although two hypothetical proteins, YbxF and YlxQ, have been proposed to be L7Ae homologs based on sequence conservation. Using purified, recombinant proteins, we show that Bacillus subtilis YbxF and YlxQ bind K-turns (K(d) ~270 nM and ~2300 nM, respectively). Crystallographic structure determination demonstrates that both YbxF and YlxQ adopt the same overall fold as L7Ae. Unlike the latter, neither bacterial protein recognizes K-loops, a structural motif that lacks the canonical helix of the K-turn. This property is shared between the bacterial and eukaryal family members. Comparison of our structure of YbxF in complex with the K-turn of the SAM-I riboswitch and previously determined structures of archaeal and eukaryal homologs bound to RNA indicates that L7Ae approaches the K-turn at a unique angle, which results in a considerably larger RNA-protein interface dominated by interactions with the noncanonical helix of the K-turn. Thus, the inability of the bacterial and eukaryal L7Ae homologs to bind K-loops probably results from their reliance on interactions with the canonical helix. The biological functions of YbxF and YlxQ remain to be determined.     

Efficient RNA 2'-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C'/D' RNPs

Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles.     

Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein

Recent investigations have identified homologs of eukaryotic box C/D small nucleolar RNAs (snoRNAs) in Archaea termed sRNAs. Archaeal homologs of the box C/D snoRNP core proteins fibrillarin and Nop56/58 have also been identified but a homolog for the eukaryotic 15.5kD snoRNP protein has not been described. Our sequence analysis of archaeal genomes reveals that the highly conserved ribosomal protein L7 exhibits extensive homology with the eukaryotic 15.5kD protein. Protein binding studies demonstrate that recombinant Methanoccocus jannaschii L7 protein binds the box C/D snoRNA core motif with the same specificity and affinity as the eukaryotic 15.5kD protein. Identical to the eukaryotic 15.5kD core protein, archaeal L7 requires a correctly folded box C/D core motif and intact boxes C and D. Mutational analysis demonstrates that critical features of the box C/D core motif essential for 15.5kD binding are also required for L7 interaction. These include stem I which juxtaposes boxes C and D, as well as the sheared G:A pairs and protruded pyrimidine nucleotide of the asymmetric bulge region. The demonstrated presence of L7Ae in the Haloarcula marismortui 50S ribosomal subunit, taken with our demonstration of the ability of L7 to bind to the box C/D snoRNA core motif, indicates that this protein serves a dual role in Archaea. L7 functioning as both an sRNP core protein and a ribosomal protein could potentially regulate and coordinate sRNP assembly with ribosome biogenesis.     

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.     

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.     

Functional requirement for symmetric assembly of archaeal box C/D small ribonucleoprotein particles

Box C/D small ribonucleoprotein particles (sRNPs) are archaeal homologs of small nucleolar ribonucleoprotein particles (snoRNPs) in eukaryotes that are responsible for site specific 2'-O-methylation of ribosomal and transfer RNAs. The function of box C/D sRNPs is characterized by step-wise assembly of three core proteins around a box C/D RNA that include fibrillarin, Nop5p, and L7Ae. The most distinct structural feature in all box C/D RNAs is the presence of two conserved box C/D motifs accompanied by often a single, and sometimes two, antisense elements located immediately upstream of either the D or D' box. Despite this asymmetric distribution of antisense elements, the bipartite feature of the box C/D motifs appears to be in pleasing agreement with a recently reported three-dimensional structure of the core protein complex between fibrillarin and Nop5p. This investigates functional implications of the symmetric features both in box C/D RNAs and in the fibrillarin-Nop5p complex. Site-directed mutagenesis was employed to generate box C/D RNAs lacking one of the two box C/D motifs and a mutant fibrillarin-Nop5p complex deficient in self-association. The ability of the mutated components to assemble and to direct methyl transfer reactions was assessed by gel mobility-shift, analytical ultracentrifugation, and in vitro catalysis studies. The results presented here suggest that, while a box C/D sRNP is capable of asymmetrical assembly, the symmetries in both the box C/D RNA and in the fibrillarin-Nop5p complex are required for efficient catalysis. These findings underscore the importance of functional assembly in methyl transfer reactions.