U6 2d Motion - Review V31 Pg 4-8 - Satter Donammis1999

Abstract

Due north ^{half dozen}-methyladenosine (m^half-dozenA) is an abundant modification in messenger RNA and noncoding RNAs that affects RNA metabolism. Methyltransferase-like poly peptide xvi (METTL16) is a recently confirmed m^sixA RNA methyltransferase that methylates U6 spliceosomal RNA and interacts with the iii′-terminal RNA triple helix of MALAT1 (metastasis-associated lung adenocarcinoma transcript one). Here, nosotros nowadays two 10-ray crystal structures of the Northward-terminal methyltransferase domain (residues 1–291) of human METTL16 (METTL16_291): an apo construction at ane.9 Å resolution and a post-catalytic Southward-adenosylhomocysteine-bound complex at two.1 Å resolution. The structures revealed a highly conserved Rossmann fold that is characteristic of Class I Southward-adenosylmethionine-dependent methyltransferases and a big, positively charged groove. This groove probable represents the RNA-binding site and it includes structural elements unique to METTL16. In-depth assay of the active site led to a model of the methyl transfer reaction catalyzed by METTL16. In dissimilarity to the major thousand⁶A methyltransferase heterodimer METTL3/METTL14, full-length METTL16 forms a homodimer and METTL16_291 exists equally a monomer based on size-exclusion chromatography. A native gel-shift assay shows that METTL16 binds to the MALAT1 RNA triple helix, merely monomeric METTL16_291 does not. Our results provide insights into the molecular structure of METTL16, which is distinct from METTL3/METTL14.

Introduction

N ^six-methyladenosine (chiliad⁶A) is the production of a dynamic and abundant modification in eukaryotic messenger RNA (mRNA) and noncoding RNAs (ncRNA)ⁱ that affects RNA stability², pre-mRNA processingⁱⁱⁱ, microRNA biogenesis⁴ and translation efficiency⁵. Approximately three to v thou⁶A sites occur in each mRNA molecule, predominantly inside 3′-untranslated regions and near terminate codons^6,vii. Nearly m⁶A sites lie within a highly conserved RRACH (R = A or M; H = A, C or U) sequence motif^1,viii and are regulated by adenosine methyltransferases ('writers'), thousand⁶A-binding proteins ('readers') and m⁶A-demethylating enzymes ('erasers')⁹.

Near 1000⁶A RNA marks are catalyzed by a heterodimeric 'writer' circuitous comprised of methyltransferase-similar protein 3 and methyltransferase-like protein xiv (METTL3/METTL14), which specifically methylates adenosine within the RRACH motif with no obvious structural preferences¹⁰. Recently, methyltransferase-like protein 16 (METTL16) was confirmed to be an thousand^{half dozen}A RNA methyltransferase that modifies U6 spliceosomal RNA^11,12 and the MAT2A mRNA encoding S-adenosylmethionine (SAM) synthase^11,13. In addition, METTL16 besides binds to ribosomal RNA, mRNA, and long ncRNAs, such every bit 10-inactive specific transcript and the 3′ triple helix of metastasis-associated lung adenocarcinoma transcript ane (MALAT1)^12,fourteen. Unlike METTL3/METTL14, METTL16-dependent m^viA marks do not occur within the RRACH sequence motif and they are found in introns and at intron-exon boundaries^11,12. Preliminary studies advise that METTL16 uses a combination of sequence and construction to recognize its RNA substrates^eleven. Interestingly, one written report has proposed that METTL16 has evolved an additional part in pre-mRNA splicing, allowing METTL16 to office as both an 1000^viA 'writer' and 'reader'¹¹. As an m⁶A 'author', METTL16 rapidly methylates the MAT2A mRNA in the presence of SAM, leading to intron retentivity followed past nuclear degradation¹¹. When SAM levels are low, prolonged occupation of METTL16 on MAT2A mRNA enhances splicing of retained intron¹¹.

METTL16 homologs are found from Escherichia coli to human and they all possess an Due north-terminal methyltransferase domain^{11,15,sixteen,17}. As a SAM-dependent methyltransferase (SAM-MTase), METTL16 is predicted to have a highly conserved Rossmann fold^eighteen. In characterized MTases, the conserved Rossmann fold is composed of alternating β strands and α helices, forming a seven-stranded β canvass that is sandwiched between clusters of α helices. In general, the SAM-bounden site is usually institute in the N-terminal segment of the β sheet, whereas the substrate-binding site lies inside the C-final segment¹⁹. Although MTases are structurally similar, they utilize a multifariousness of mechanisms to specifically recognize their targets, such as oligomerization and unique structural elements. These unique structural elements include variable loop regions within the Rossmann fold and auxiliary domains within and/or flanking the Rossmann fold^20,21,22.

In this work, we nowadays the structural analysis of homo METTL16 based on ii 10-ray crystal structures of the N-last MTase domain (residues 1–291) of human METTL16 (METTL16_291): an apo form at 1.nine Å resolution and a post-catalytic complex with South-adenosylhomocysteine (SAH) at 2.1 Å resolution. We analyzed the salient MTase features of METTL16_291 based on characterized SAM-MTases; this assay highlights the unique attributes of METTL16_291 and contrasts it to the structural studies of METTL3/METTL14. Moreover, we investigated the oligomeric land of METTL16 and its power to demark to the MALAT1 RNA triple helix.

Results and Discussion

METTL16_291 is a SAM-dependent MTase with a conserved Rossmann fold

In general, m⁶A RNA MTases bind to SAM and RNA, transfer a methyl group from SAM to adenosine, and then the methylated RNA and SAH products dissociate from the MTase (Fig. 1A). To provide structural insights into the methyltransferase reaction catalyzed by the MTase domain of METTL16, nosotros solved X-ray crystal structures of apo and SAH-bound METTL16_291 (METTL16_291/SAH, Fig. ane and Table 1). These structures confirmed that METTL16 is a Form I SAM-MTase that uses a conserved Rossmann fold to demark the SAH product and probable SAM, the methyl-donor substrate (Figs. 1B–Eastward and S1). The conserved secondary structural elements of METTL16_291 are numbered co-ordinate to the canonical SAM-MTase fold¹⁹ (Fig. 1C,D). The conserved SAM-MTase cadre (residues 79–288) of METTL16_291 possesses a generally parallel, 7-stranded β sheet (β1–β7) organized as '3214576', with β7 existence antiparallel (Fig. 1B–D). The β canvass appears to be stabilized by a disulfide bridge between C183 and C247, thereby linking the β4 and β5 strands. This disulfide span is nowadays at approximately 40% occupancy in the apo structure and is absent in the SAH-bound complex, likely because of radiation damage and/or the presence of Tris(two-carboxyethyl)phosphine hydrochloride (TCEP) in the protein buffer. Including TCEP was necessary to prevent protein precipitation. The '3214576' β canvass is packed betwixt two clusters of α and 3₁₀ (η) helices (Fig. 1C,D). Although the MTase domain of METTL16 has a canonical Course I MTase structure, METTL16_291 has several unique structural regions: residues 1–78 that precede the Rossmann fold also every bit residues 95–97 (ηZ) and 188–222 (a putative loop containing disordered residues 188–214) within the Rossmann fold (Fig. 1C,D). Altogether, these structural elements unique to METTL16 likely contribute to its RNA substrate specificity that is distinctly different from the METTL3/METTL14 complex^{half-dozen,seven,x,11,12,22,23,24}.

Table one Information collection and refinement statistics.

Total size tabular array

Cofactor-binding site is defined by extensive network of noncovalent interactions

As an grand⁶A RNA MTase, METTL16 interacts with iii different ligands: SAM, SAH and RNA (Fig. 1A)^xi,12. To report ligand bounden, we soaked METTL16_291 crystals with SAM, SAH, adenosine 5′-monophosphate (a mimic of the methyl-acceptor adenosine) and diverse RNAs but obtained merely a SAH-bound complex after soaking crystals with SAM. The presence of SAH, rather than SAM, was determined based on calculations of electron density maps (Fig. 1E) using SAM and SAH each equally models (see Methods).

Our crystal structure shows that METTL16_291 binds SAH, and probable SAM as well, inside a deep pocket of the Rossmann fold (Figs. 1D,E and 2A). Multiple hydrogen bonds ensure the proper positioning of the adenine, ribose and homocysteine moieties of SAH (Fig. 2A). The adenine moiety of SAH is recognized past the main chain of T164 in addition to a hydrophobic pocket created by the side chains of I109, V134, V160, L165 and F227 (Fig. 2). The 2′- and 3′-hydroxyl groups of the ribose are recognized via hydrogen bonding to E133, a universally conserved residue in SAM-MTases (Figs. 2A and S1)¹⁹. The sulfur atom of SAH probable hypervalently interacts²⁵ with the courage carbonyl oxygen atoms of N184 and P186, which are part of the conserved catalytic motif (see below). R82 forms a salt bridge with the carboxyl group of SAH. Moreover, half-dozen water molecules mediate hydrogen-bonding interactions between SAH and D108, I109, T111, S114, Y117, T132, Q162 and R230 (Fig. 2A). Lastly, the nucleotide-binding site includes the conserved GXG motif (G110-T111-G112) that is characteristic of the SAM-MTase core (Fig. S1)^nineteen,26. This motif shapes the cavity between adenosine and the homocysteine moieties merely only T111 interacts with SAH via a water-mediated hydrogen bond (Fig. 2A).

Next, we compared the SAH-bounden sites of our METTL16_291/SAH structure and an unpublished 10-ray crystal structure of METTL16 containing residues 40–291 (METTL16_40–291/SAH, Fig. 2B, PDB ID: 2h00, Structural Genomics Consortium²⁷). From this comparative analysis, we discovered that most METTL16-SAH interactions overlap (Fig. 2C). However, the homocysteine moiety in METTL16_40–291, particularly the location of the sulfur atom (shifted by ii.three Å in comparison to METTL16_291), is reoriented due to the dissimilar conformations of R82 and N184 (Fig. 2C). Farther assay of SAH-bound METTL16_291 with 10-ray crystal structures of other Class I SAM-MTases (PDB ID: 1boo²⁸, 1qan²⁹, 1eiz³⁰, 1hnn³¹, and 1khh³²) indicates that the sulfur cantlet location of SAH in our circuitous is unique, which may be a crystallization antiquity resulting from an interaction between R82 and E217 of a symmetrically related molecule in the crystal lattice. Importantly, the conformation of SAH in METTL16_40–291/SAH, as preserved in other SAM-MTases, is properly oriented after a productive methyl transfer reaction. Thus, these structural snapshots suggest that METTL16_40–291/SAH represents a post-catalytic land that likely occurs before the country captured in METTL16_291/SAH.

Structural comparisons of apo and SAH-leap METTL16_291 structures revealed that the cofactor-binding site in the apo structure is occupied by four water molecules, which serve every bit placeholders for the following heteroatoms: (i) amino and carboxyl groups of the homocysteine moiety, (two) iii′-hydroxyl of the ribose moiety and (iii) N1 of adenine. Altogether, our structures indicate that METTL16 uses an extensive network of noncovalent interactions to facilitate bounden of SAH and likely SAM as well.

METTL16 has a large, positively charged groove to collaborate with RNA

To identify a putative RNA-bounden site, nosotros inspected the surface electrostatic potential of METTL16_291/SAH using the PDB2PQR server³³ and institute a positively charged groove covering an expanse of ~2000 Å², which could interact favorably with the negatively charged phosphate backbone of RNA (Fig. 3A). Residues lining the groove are highly conserved amongst Chordata METTL16 homologs (Fig. S1), thereby supporting the putative functional importance of this groove. The positively charged surface of METTL16 includes K5, K14, R41 and K47 in the unique N-terminal region; R82, K251, K252, R279 and R282 in the conserved Rossmann fold; and dipoles of the helices αNA, αNC, αZ, αηD and ηαE (Figs. 1D and 3B). The N-last residues ane–78 most probable contribute to the unique RNA substrate specificity of METTL16. Interestingly, residues K47 and R279 protrude from the surface to grade a hook-like structure broad enough (~eight Å) to arrange a phosphate group in the RNA courage (Fig. 3A).

With our current structures of METTL16, information technology is not possible to predict how METTL16 recognizes its RNA substrates for three reasons. First, METTL16 may undergo conformational changes upon RNA binding, as observed for the 5-methylcytosine DNA methyltransferase from Haemophilus haemolyticus ³⁴. Second, the C-terminal domain of METTL16 (residues 292–562) is missing in our structure and may also contribute to substrate recognition. 3rd, the RNA substrate specificity of METTL16 is non yet clear. The m⁶A sites in U6 snRNA and MAT2A mRNA, ii confirmed substrates, reside in unmarried-stranded bulges flanked by double-stranded RNA^35,36. The active site of METTL16_291/SAH tin easily arrange unmarried-stranded RNA within a ~37 × 18 Å groove (Fig. 3A). Moreover, the conserved residues of METTL16 essential for catalysis, ₁₈₄NPPF₁₈₇, are located between the SAM/SAH-bounden site and the positively charged groove, suggesting that this region is indeed the agile site of METTL16 (Fig. 3A). Thus, the putative RNA-binding site of METTL16 is compatible with the canonical SAM-MTase architecture, whereby the RNA substrate-bounden site is located inside the C-terminal segment of the parallel β canvas and the SAM-bounden site is situated within the N-terminal segment of the parallel β sheet (Fig. 1C)¹⁹.

METTL16 uses a conserved mechanism to catalyze methyl transfer

The full general reaction machinery catalyzed by thou⁶A MTases has been established^37,38. These enzymes contain a conserved [DNSH]PP[YFW] motif³⁸, which corresponds to ₁₈₄NPPF₁₈₇ in METTL16 (Figs. 4A and S1). Contempo studies of METTL16_291 confirmed that conserved residues P185, P186 and F187 are essential for m^half-dozenA methylation considering METTL16_291 PP185/186AA and F187G mutants failed to methylate U6 and MAT2A RNA substrates in vitro ^eleven. Here, we propose a model of methyl transfer from SAM to the N6 amino group of adenosine past superposing the SAH-bound METTL16_40–291 structure with an thousand^sixA Deoxyribonucleic acid MTase from E. coli, EcoP15l (PDB ID: 4zcf, chain B), in complex with an extrahelical adenosine acceptor (Fig. 4B)³⁹. The protein chains of METTL16_40–291 and EcoP15l overlap with an rmsd value of 1.8 Å across 36 Cα atoms, and the acceptor 2′-deoxyadenosine appears poised for methyl transfer in the active site of METTL16_40–291/SAH modeled with SAM. Like other 1000^viA MTases^21,37,38, METTL16 probable uses the Oδ atom of N184 and carbonyl oxygen of P185 to negatively polarize the N6 amino grouping of adenosine via hydrogen bonding (Fig. 4A,B). As a result, the amino group becomes primed for transfer of the methyl group from SAM via an Southward_N2 machinery. The resulting Due north ⁶-methylammonium adenosine cation could exist stabilized past the same atoms that hydrogen bond to the amino group.

In general, chiliad⁶A MTases use the effluvious residues Y, F or Westward in the [DNSH]PP[YFW] motif to hold the extrahelical, 180°-rotated nucleobase adenine in place past π-π stacking^37,38,39. In our model, the methyl-acceptor adenosine is situated in a hydrophobic environment formed by L75 and F187, but F187 is not in an optimal conformation to stack with adenosine (Fig. 4B). However, with the proper substrates, METTL16 could likely reposition F187 for optimal π-π stacking interactions considering ₁₈₄NPPFF₁₈₈ undergoes a conformation rearrangement upon cofactor bounden (Fig. 4C). Comparison of the apo METTL16_291, SAH-leap METTL16_291, and SAH-leap METTL16_40–291 structures revealed significantly different orientations of the ₁₈₄NPPFF₁₈₈ motif. Notably, apo METTL16_291 and SAH-bound METTL16_40–291 show a trans-conformation betwixt P185 and P186 whereas the SAH-jump METTL16_291 complex shows a cis-conformation (Fig. 4C). Additionally, the ₁₈₄NPPF₁₈₇ residues are adjacent to a disordered region spanning 189–213 in the apo structure and 188–214 in the SAH-jump complex; these residues could non be modeled due to a lack of electron density. This disordered segment is one of the elements that distinguishes the Rossmann fold of METTL16 from other SAM-MTases (Fig. 1C,D). Hence, it may play an important office in RNA substrate recognition and/or orienting molecules for catalysis.

METTL16 is a homodimeric RNA MTase

Most Class I SAM-MTases are monomeric, although some MTases grade dimers or tetramers to recognize their substrates, to assemble their agile sites or to facilitate catalysis^21,37. For case, the heterodimeric METTL3/METTL14 complex forms a putative RNA-bounden site at the METTL3-METTL14 interface^22,23,24 and a METTL16 ortholog in Caenorhabditis elegans exists as a homodimer⁴⁰. Therefore, we used size-exclusion chromatography (SEC) to examine the oligomerization states of both human being METTL16 and METTL16_291. Our SEC data signal that METTL16 exists as a homodimer considering it elutes at a volume equivalent to a molecular weight of ~103 kDa, which is most twice the size of a unmarried METTL16 polypeptide concatenation (theoretical MW 63.7 kDa) (Fig. 5A). Furthermore, its slower migration suggests that homodimeric METTL16 has a non-globular conformation. To further investigate the oligomerization of METTL16, nosotros used SEC followed past small-angle X-ray scattering (SEC-SAXS). SEC-SAXS is a technique that uses X-ray scattering data of macromolecules in solution to determine their molecular weights, which is then used to deduce the oligomeric state of a complex eluting from the SEC column. Hither, our SEC-SAXS analysis confirmed the homodimeric country of METTL16, determining a molecular weight of 125 kDa (theoretical MW 127.4 kDa) (Fig. S2A–C). In contrast, METTL16_291, whose theoretical molecular weight is 33.3 kDa, is a monomer, eluting from SEC at a volume equivalent to ~31 kDa (Fig. 5A). These results propose that the C-terminal domain of human METTL16, which contains an evolutionarily variable region (residues 402–498) predicted to be disordered past MobiDB⁴¹, is required for oligomerization (Figs. 1B and S1). This finding is consequent with the oligomerization studies of the METTL16 ortholog in C. elegans ^xl. Notably, homodimerization of METTL16 does not appear to be required for RNA binding and MTase activity because METTL16_291 methylates U6 and MAT2A RNA substrates in vitro ¹¹.

Recently, METTL16 was shown to interact with the RNA triple helix at the three′ end of MALAT1¹⁴. Therefore, we used a native electrophoretic mobility shift analysis (EMSA) to determine if METTL16 and METTL16_291 can interact with the MALAT1 RNA triple helix in vitro. Our results testify that monomeric METTL16_291 (1 μM) does not bind to the MALAT1 RNA triple helix but METTL16 (0.5 µM dimer) does bind (Fig. 5B). Next, we used SEC and SEC-SAXS to examine the stoichiometry of the METTL16/MALAT1 RNA triple helix circuitous. SEC data showed that MALAT1 elutes at a volume equivalent to a molecular weight of ~56 kDa (Fig. 5A) whereas SEC-SAXS determined a molecular weight of 36 kDa (theoretical MW 30.2 kDa) (Fig. S2D–F). The apparent college molecular weight of MALAT1 RNA is consistent with previous SEC studies showing that RNAs carry every bit globular proteins upwards to five fold larger than their theoretical molecular weights⁴². When METTL16 is mixed with the MALAT1 RNA triple helix at a 1:1 molar ratio, the SEC profile showed two peaks: one peak respective to the METTL16/MALAT1 RNA circuitous at ~188 kDa and i peak respective to unbound RNA at ~51 kDa (Fig. 5A). SEC-SAXS was used to examine the METTL16/MALAT1 RNA peak, and these results revealed a molecular weight of 155 kDa (theoretical MW 157 kDa), indicating that the METTL16 homodimer interacts with one molecule of MALAT1 RNA (Fig. S2G–I). Thus, these EMSA, SEC and SEC-SAXS results propose that METTL16 may utilize different mechanisms to interact with unlike RNAs considering just dimeric METTL16 interacts with the MALAT1 RNA triple helix but monomeric METTL16_291 binds and methylates both U6 and MAT2A RNAs^eleven.

Structural comparison of METTL16_291 and METTL3/METTL14

METTL16 is the second catalytically active m^viA mRNA MTase to be validated in humans^xi. The only other m^{half dozen}A mRNA MTase currently known is the heterodimeric METTL3/METTL14 complex, whereby METTL3 has MTase activity and METTL14 functions as a scaffold to bind RNA^22,23,24. Therefore, nosotros superposed the 10-ray crystal structures of human METTL3 (PDB ID: 5il2, residues 369–570) and human METTL14 (PDB ID: 5il2, residues 117–420) in complex with SAH²² onto SAH-bound METTL16_291. This comparative structural analysis revealed that METTL16_291 is like to merely METTL3 (rmsd 2.i Å across 40 Cα atom pairs) and not METTL14 (Fig. half-dozen). In METTL3, its conserved Rossmann fold includes 4 α helices, three iii₁₀ helices and an eight-stranded parallel β canvas organized as '18723546', with the β5 strand being antiparallel²² (Fig. 6A,B). This β-sheet organization is distinctly different from that of METTL16_291 ('3214576' in Fig. 6B), which explains the lack of sequence similarity between METTL16 and METTL3. Withal, the 3D spatial positioning of β strands and α helices within the Rossmann fold is similar except for the unique β6 strand and α4 helix of METTL3 and the unique αB helix of METTL16 (Fig. 6B). The SAH-binding sites in METTL3 and METTL16_291 are conserved in the Rossmann fold, although balance identities are dissimilar. Nonetheless, METTL16 and METTL3 form noncovalent interactions with the backbone atoms of homocysteine and N6, N1, 2′-hydroxyl and 3′-hydroxyl groups of adenosine in SAH.

The structural ground of RNA recognition by METTL16 and METTL3/METTL14 remains unclear. However, it has been proposed that the gate loops of METTL3/METTL14 play a role in RNA substrate recognition²². These gate loops map to the same face of the protein construction as the positively charged groove of METTL16_291 (Fig. 6C). More precisely, gate loop i of METTL3 (residues 395–410) corresponds to residues 184–222 in METTL16, a predicted loop that includes disordered residues 188–214 (Fig. 6B). These regions include the conserved catalytic residues: ₃₉₅DPPW₃₉₈ in METTL3 and ₁₈₄NPPF₁₈₇ in METTL16. The structural counterpart of gate loop 2 in METTL3 (residues 507–515) is the loop betwixt αNC and αZ as well every bit N-concluding office of αZ in METTL16_291 (Figs. 1C,D and 6B). Both of these regions contribute to the positively charged groove (Fig. 6C). Interestingly, the long interface loop of METTL3 (residues 462–479) that interacts with METTL14 corresponds to the short β hairpin betwixt strands β6 and β7, which contains R279 involved in the K47-R279 claw-like structure, in METTL16 (Figs. 3A and 6B). Thus, this comparative analysis shows that structural elements unique to METTL3 and METTL16 are located primarily in the RNA-binding site, providing a structural basis for why METTL3/METTL14 and METTL16 recognize different RNA substrates^{6,vii,10,11,12,22}.

Role of METTL16 as an m⁶A RNA MTase

mRNA modifications, especially one thousand⁶A, accept been intensely studied in the past few years. Much of the work has focused on METTL3/METTL14, although information technology is now clear that METTL16 too plays a key role in m⁶A biology^11,12,13. Recent studies suggest that METTL16 does not follow trends more often than not associated with yard^sixA: (i) RNA targets do not have a RRACH motif and (two) bulk of METTL16-dependent thou⁶A marks are found in introns^1,8,11,12,43. These findings might not be that surprising because METTL16 knockdown accounts for a ~20% loss of the m^half-dozenA methylome in 293A-TOA cells¹¹. Notwithstanding, METTL16, which is distributed throughout the nucleus of HeLa cells, interacts with a broad range of RNAs, including long ncRNA, mRNA, miRNA, rRNA, snRNA and snoRNA^11,12,14. Validated m^{half dozen}A targets include U6 snRNA, whose methyl mark at A43 is apparently essential for spliceosome activity, and MAT2A mRNA; both RNAs are methylated by METTL16 in a UACm^six_AGA sequence^11,12,13. METTL16 also interacts with the MALAT1 RNA triple helix, which is 13 base pairs from a weak m⁶A indicate most A8290 in a ACAm⁶_ACA sequence^xiv,44. Thus, it will be interesting to decide if the MALAT1 RNA triple helix itself or side by side regions are substrates of METTL16 considering MALAT1 is a cancer-promoting long ncRNA⁴⁵. Furthermore, our studies bear witness that full-length METTL16 is required to bind to the MALAT1 RNA triple helix, suggesting that the C-terminal domain expands the RNA interactome of METTL16. Two regions of the C-terminal domain of METTL16 (residues 289–399 and 514–562) are conserved in vertebrates¹¹ while the N-last MTase domain is conserved from East. coli through human being (Figs. 1B and S1). METTL16_291 shares 31% identity and 50% similarity to a METTL16 homolog in E. coli: RlmF (or ybiN), which methylates A1618 in 23 South rRNA¹⁵. Both METTL16 and RlmF can install chiliad⁶A marks within the ACm⁶_AGR sequence^{xi,thirteen,xv}. Man METTL16 interacts with eighteen South and 28 Southward rRNAs, which contain m⁶A marks at A1832 and A4220, respectively^{12,14,46,47,48}. These marks occur within a GUAm⁶_ACR sequence, which would appear to be a suboptimal substrate for METTL3/METTL14 and maybe METTL16. Interestingly, the Am^six_ACR motif found in rRNA besides appears at A8290 in MALAT1 RNA. Some other factor that may influence RNA substrate recognition and MTase action is protein cofactors. Thus far, La protein, La-related protein 7, and methylphosphate capping enzyme take been identified as binding partners of METTL16 in a U6 snRNA-dependent manner¹². Thus, more cellular, biochemical, and structural studies of METTL16 are necessary to elucidate its part at the molecular level.

Conclusions

Our work expands the structural knowledge of human thousand^half-dozenA RNA MTases by analyzing the ligand-binding sites and proposing a structural model of methyl transfer catalyzed by METTL16. Interestingly, METTL16 has an extensive, positively charged groove to demark RNA and this groove is largely comprised of structural elements unique to METTL16, suggesting this surface area may contribute to the RNA substrate specificity of METTL16 that is distinctly different from METTL3/METTL14. Moreover, our SEC and SEC-SAXS information show that METTL16 is a homodimer; yet, the Northward-terminal domain of METTL16 is a monomer, indicating that the C-terminal domain facilitates protein dimerization. Homodimeric METTL16 is required to bind to the MALAT1 RNA triple helix while monomeric METTL16_291 is sufficient to methylate U6 and MAT2A RNAs¹¹. Hopefully, future studies will reveal the biochemical and structural ground of the unique RNA substrate specificity of METTL16 and its roles every bit an m⁶A 'writer' and 'reader'.

Methods

Cloning, overexpression and purification of METTL16_291 and METTL16

The sequences encoding human METTL16_291 (residues one–291) and METTL16 (residues 1–562) [UniProt ID: Q86W50] were amplified by PCR using a pcDNA3-METTL16 plasmid and the following primers: Frontward: 5′-TACTTCCAATCCAATGCCATGGCTCTGAGTAAATCAATGCATGCAA-3′ and Contrary: v′-TTATCCACTTCCAATGTTACTAATCATAAAAACTCCAAGCTAAGGCC-3′ for METTL16_291; Forward: 5′-TACTTCCAATCCAATGCCATGGCTCTGAGTAAATCAATGCATGCAA-three′ and Reverse: 5′-TTATCCACTTCCAATGTTACTAGTTAACTGCAACAAGCCTGAAAATTTG-3′ for METTL16. Then, the Deoxyribonucleic acid insert was incorporated into the pMCSG68 vector (Midwest Center for Structural Genomics) using a ligase-independent cloning method⁴⁹. The expressed METTL16_291 and METTL16 proteins have an N-final His_half-dozen-tag followed by a Tobacco Etch Virus (TEV) protease cleavage site and a Ser-Asn-Ala linker, which is encoded in the pMCSG68 vector. The sequence of the factor was confirmed by DNA sequencing (University of Notre Dame Genomics Facility).

Protein overexpression was carried out in BL21 Gold E. coli (Agilent Technologies) cells grown in 2 L LB media supplemented with 150 μg/ml ampicillin. The leaner were cultured with shaking at 190 RPM at 37 °C until the OD₆₀₀ exceeded 1.0 (~4 h). After, cultures were chilled to eighteen °C, and protein expression was induced by the add-on of 0.5 mM isopropyl-D-thiogalactopyranoside for 18 h. Cells were harvested by centrifugation at 5,000 x g for xv min at 4 °C, resuspended in ice-cold bounden buffer [fifty mM HEPES-NaOH pH 7.5 at RT, 500 mM NaCl, 20 mM imidazole and 1 mM tris(two-carboxyethyl)phosphine (TCEP)] and stored at −80 °C.

Thawed cells were disrupted by sonication (iv min total of probe working time), using 4-s bursts with 26-s intervals for cooling in an water ice/water bathroom. Lysate was cleared by centrifugation at 26,000 x g for xl min at 4 °C. The supernatant was transferred to a 50-ml column packed with 3 ml of pre-done HisTrap HP resin (GE Healthcare) and incubated for 5 min at 4 °C. The column was connected to a Vac-Man Laboratory Vacuum Manifold (Promega) to wash the poly peptide-bound resin half dozen times with 50 ml binding buffer for each wash. The His₆-tagged protein was eluted by gravity with 20 ml elution buffer (l mM HEPES-NaOH pH seven.v at RT, 500 mM NaCl, 400 mM imidazole and one mM TCEP). The imidazole concentration was lowered to 20 mM and the His₆-tag was cleaved with TEV protease (last concentration 0.1 mg/mL) during dialysis for 18 h at 4 °C. The sample was reapplied onto a HisTrap HP resin to remove both the broken His_vi-tag and the His_{half dozen}-tagged TEV protease. Period-through was collected and concentrated to ii.0 ml using Amicon ultra centrifugal filters (30-kDa cutoff, Millipore). The poly peptide was further purified by SEC (HiLoad 16/600 Superdex 200 pg, GE Healthcare) using size exclusion buffer (SEB) containing 25 mM HEPES pH 7.5 at RT, 100 mM KCl, 50 mM NaCl and 1 mM TCEP. The peak fractions of METTL16_291 and METTL16 were pooled and full-bodied to 27 mg/ml and 10 mg/ml, respectively. A₂₈₀ values were measured and molarity calculated using an extinction coefficient of 43890 and 63370 L/(mole·cm) for METTL16_291 and METTL16, respectively.

Crystallization and diffraction data collection

Crystals grew at 19 °C using the hanging-drop method. The crystallization driblet independent 4 µl METTL16_291 (27 mg/ml) and two µl of reservoir solution, which independent ane.3 1000 K₂HPO_four, 45 mM NaH₂PO₄, pH 8.five. Crystals measuring approximately 0.3 × 0.3 × 0.2 mm appeared after iii days. To obtain the SAH-bound METTL16_291 circuitous, 0.2 µl of 200 mM SAM solution (Sigma-Aldrich, buffered in 50 mM HEPES pH seven.5) was added to the drib with mature crystals. Crystals were cryoprotected by soaking in crystallization solution supplemented with 25% ethylene glycol, vitrified in liquid nitrogen and stored until data collection. Diffraction data were collected under cryocooled conditions (100 K) at the Avant-garde Photon Source, Argonne National Laboratory, on beamline 22-ID. XDS⁵⁰ package was used for information reduction. The statistics of the information collection and processing are summarized in Table 1.

Decision and refinement of the crystal structures

The crystal structures of METTL16_291 were solved past molecular replacement using Phaser⁵¹ and the X-ray crystal structure of homo methyltransferase 10 domain containing protein (94% identity, PDB ID: 2h00²⁷). ARP/wARP⁵² was used to build the initial model, which afterwards was placed inside the unit prison cell with the ACHESYM server⁵³. Transmission plumbing fixtures in the electron density maps was completed in COOT⁵⁴ with iterative rounds of model refinement in Refmac⁵⁵. Ten and four TLS^56,57 groups were added for apo and SAH-jump METTL16_291, respectively, as recommended by the TLSMD server⁵⁸. The refinement statistics are listed in Table 1. Our final models include residues three–291 (missing 189–213) in apo METTL16_291 and 5–291 (missing 188–214) for SAH-bound METTL16_291. The presence of SAH, rather than SAM, was determined based on the electron density maps calculated for refined models with either of the two ligands. No peak of positive density appeared at the position corresponding to the methyl group when SAH was placed in the model, whereas for SAM, the methyl group lied in a acme of negative density. A sodium cation in the apo structure was modeled on the basis of octahedral coordination sphere (with four out of half-dozen electron pair donors) and the interatomic distances that refined to values below 2.4 Å. 3 and four ethylene glycol molecules were modeled in the apo and SAH-bound METTL16_291 structures, respectively.

Belittling SEC

The 94-nt MALAT1 RNA triple helix 5′-GGAAGGUUUUUCUUUUCCUGAGAAAACAACACGUAUUGUUUUCUCAGGUUUUGCUUUUUGGCCUUUUUCUAGCUUAAAAAAAAAAAAAGCAAAA-3′ was generated using the pHDV-MALAT1 ENE + A WT plasmid equally described previously^fourteen,59. All samples (METTL16_291, METTL16, MALAT1 RNA triple helix and METTL16/MALAT1 RNA triple helix) were diluted to ~21 µM in 100 µl full volume of SEB buffer supplemented with 1 mM MgCl₂. RNA was denaturated at 95 °C for ane min, snap-cooled on ice for five min and incubated at room temperature for ane h. METTL16 and the MALAT1 RNA triple helix were mixed at a one:1 ratio of 21 μM:21 μM and incubated at room temperature for 45 min prior to injection. Samples were injected onto a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with SEB buffer supplemented with i mM MgCl_ii and resolved at a menses rate of 0.vi ml/min. Thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa)) were used every bit standards (Gel Filtration Standard, BioRad). Molecular weights of METTL16_291, METTL16, MALAT1 RNA triple helix and METTL16/MALAT1 RNA triple helix circuitous were calculated using a standard curve.

Small-angle 10-ray handful (SAXS) measurements

Solution SAXS data were collected at the Advanced Photon Source BioCat, 18-ID beamline in the SEC-SAXS pipeline^threescore. Samples pre-equilibrated in SEB buffer supplemented with ane mM MgCl_ii were injected onto the SEC column at the post-obit concentrations: xxx μM METTL16, 25 μM MALAT1 RNA and METTL16 + MALAT1 RNA complex at a 2:1 ratio of 30 μM:15 μM. Data were recorded at room temperature on a PILATUS 3 ane M detector with 0.5-s exposures every 3 s at one.0 Å wavelength. Sample jail cell-to-detector distance was 3.v m, and the data range was 0.0163 Å⁻¹ to 0.3666 Å^−ane (q = 4π sin θ/λ, where 2θ is the scattering angle, and λ is the Ten-ray wavelength). Baseline scattering was averaged from data points measured before and later the protein peak, whereas sample frames corresponding to the elution peak were averaged to maximize the point-to-noise ratio. Data were candy using the BioCAT beamline pipeline, which is based on ATSAS package⁶¹. Guinier assay was performed in PRIMUS ⁶² and GNOM ⁶³ for calculation of the radius of gyration (Rg) and the pair distribution function, P(r). Molecular masses are based on the calculation of low-resolution ab initio models with the use of DAMMIF ⁶⁴, DAMAVER ⁶⁵, DAMMIN ⁶⁶, and DAMFILT.

Electrophoretic mobility shift assay (EMSA)

The MALAT1 RNA triple helix was 5′-end radiolabeled with [γ-³²P]ATP equally described previously¹⁴. Immediately before incubating RNA with protein, RNA was denaturated at 95 °C for 1 min, snap-cooled on ice for 5 min, and incubated at room temperature for 1 h. Then, two nM 5′-[³²P]-labeled MALAT1 RNA was mixed with 1 μM METTL16_291 or METTL16 in binding buffer (25 mM Tris pH 7.five, 25 mM NaCl, 150 mM KCl, 1 mM MgCl_ii, ane mM DTT, 0.v mg/ml tRNA, 1U/μl RNase Inhibitor and 8% glycerol) at room temperature for 45 min. The reactions were resolved using five–6% native polyacrylamide gel (nineteen:ane acrylamide:bisacrylamide, one × Tris-Borate buffer (TB), 1 mM MgCl₂) and running buffer (0.75 × TB, 1 mM MgCl₂). Electrophoresis was run under an electrical field of 8 V cm⁻¹ for two.5 h. Gels were visualized on phosphor screen and scanned using an Amersham Typhoon IP Biomolecular Imager (GE Healthcare).

Other software used

Disordered region of METTL16 was calculated past MobiDB server⁴¹. Molecular illustrations were created using UCSF Chimera⁶⁷, which also calculated rmsd values across pairs of Cα atoms inside 3 Å radius for all superposition analyses. Surface electrostatic potential was calculated using the PDB2PQR server³³. The amino acrid sequences of METTL16 from 394 chordates were aligned using ClustalX2⁶⁸ and conservation calculated using the ConSurf server⁶⁹.

Accession numbers

Coordinates and construction factors were deposited in the Protein Data Bank (PDB): apo METTL16_291, PDB ID: 6b91; METTL16_291/SAH complex, PDB ID: 6b92.

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Acknowledgements

This project was supported past NIH Grant R00GM111430, the Clare Boothe Luce Program of the Henry Luce Foundation, startup funds from the Academy of Notre Dame, and partially by the Intramural Research Plan of the NCI Center for Cancer Research. Diffraction data were collected at the SER-True cat beamline 22-ID at the Advanced Photon Source, Argonne National Laboratory, supported by the U.South. Department of Energy, Office of Scientific discipline, Office of Bones Energy Sciences under Contract Westward-31–109-Eng-38. This inquiry used resources of the Advanced Photon Source, a U.Due south. Section of Energy (DOE) Part of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by grant nine P41 GM103622 from the NIGMS. Use of the Pilatus 3 1 M detector was provided by grant 1S10OD018090-01 from NIGMS. The authors are thankful to Srinivas Chakravarthy (Biophysics Collaborative Access Team, Argonne National Laboratory) for his help during SAXS data drove and analysis, Patricia Clark (University of Notre Dame) for utilise of sonicator and Phillip McCown (Academy of Notre Dame) for a protein sequence alignment file of METTL16 homologs.

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Department of Chemical science and Biochemistry, Academy of Notre Dame, Notre Matriarch, IN, 46556, USA

Agnieszka Ruszkowska & Jessica A. Brown
Synchrotron Radiation Enquiry Department of MCL, National Cancer Institute, Argonne, IL, 60439, USA

Milosz Ruszkowski & Zbigniew Dauter

Contributions

A.R. and J.A.B. designed experiments; A.R. performed the experiments; A.R., Thou.R., Z.D., and J.A.B. analyzed the results; A.R., M.R., and J.A.B. wrote the manuscript.

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Correspondence to Jessica A. Brown.

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Ruszkowska, A., Ruszkowski, M., Dauter, Z. et al. Structural insights into the RNA methyltransferase domain of METTL16. Sci Rep 8, 5311 (2018). https://doi.org/x.1038/s41598-018-23608-8

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Received: 12 December 2017
Accustomed: 16 March 2018
Published: 28 March 2018
DOI : https://doi.org/10.1038/s41598-018-23608-eight

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