The latest article published by Nature of Wuhan University: collaborative research results of tRNA methyltransferase

With the establishment and development of the epitranscriptome theory, more and more studies have found that RNA modification plays a crucial role in gene expression regulation, which in turn affects a variety of biological processes
.
There are more than 170 post-transcriptional chemical modifications in the RNA family, and tRNA is one of the most advanced modifications that affect its biological functions such as maturation, stability, and translation1-3
.
N7-methylguanosine (m7G) is one of the most abundant chemical modifications on tRNA, and its methyltransferase METTL1-WDR4 complex has been shown to be associated with the initiation and regulation of diseases such as a variety of cancers4-8
。 METTL1-WDR4-mediated m7G modifications also provide new targets for cancer therapy, but the lack of accurate structural information and little understanding of the molecular mechanisms of how the modified enzyme recognizes its substrates and how its activity is regulated have largely hindered the design and development
of small molecule inhibitor drugs targeting its activity.

On January 4, Professor Longfei Wang's group from the School of Pharmacy of Wuhan University (co-first author) and Richard Gregory's group at Harvard Medical School and Boston Children's Hospital (postdoctoral fellow Jiazhi Li as the first author) published a report on Nature online entitled Structural basis of regulated m7G tRNA modification by METTL1–WDR4 Structural basis of m7G modification of tRNA by METTL1-WDR4"
.

The crystal structure of the human-derived METTL1-WDR4 complex was obtained for the first time, and the complex structure of METTL1-WDR4 with different tRNA substrates was resolved by cryo-EM, including METTL1-WDR4-tRNA Phe-SAH (naturally derived yeast tRNA Phe) and METTL1-WDR4-tRNA Val ( Electron microscopic structure
of chemically synthesized unmodified human tRNAVal) complex.
Although the source, sequence, and modification state of the tRNA of the two substrates are different, METTL1-WDR4 binds the tRNA substrate using a monolithic structure similar to a "sailboat" (Figure 1).

Based on structural information and verified by a series of biochemical, cellular and NMR experiments, the research group revealed the working mechanism model of METTL1-WDR4 to recognize substrate tRNA, found that the N-terminal of METTL1 participates in the construction of active pockets, and inhibits its methyltransferase activity
through steric hindrance effect triggered by phosphorylation.

Figure 1.
The overall structure of METTL1-WDR4-tRNAPhe

Previous studies have reported that mutations in human WDR4 (R170 site) induce microcephaly primitive dwarfism9,10
.
Our research team found that R170 is located at the B3-B4 domain interaction interface of WDR4, and the mutation of this site will reduce the stability of scaffold and affect the activity
of METTL1.
This is consistent with the previously reported results of reduced tRNA m7G modification levels in patients with primitive dwarfism of the microcephaly type, and also provides a theoretical basis
for understanding the pathogenesis and potential treatment of such diseases.

The team also captured the local unwinding of tRNA and the conformational changes of METTL1 when binding to tRNA substrates, and found that METTL1 grabbed both sides
of the tRNA's variable-loop domain through its two highly conserved helix domains.
With the assistance of WDR4, the G46 to be modified is placed in the active pocket area and the substrate is identified (Figure 2f-k).

Figure 2.
METTL1-WDR4 recognizes substrate tRNA

a-e, WDR4 as scaffold-assisted METTL1 binding substrates

f.
Structural superposition of METTL1-WDR4-tRNAPhe two states

g-j.
METTL1 recognizes both ends of the tRNAvariable loop

k.
Cartoon model of METTL1-WDR4 recognition of substrate tRNA

The study reported that the 27th Serine of METTL1 can be phosphorylated by AKT, and phosphorylation of Ser27 leads to the inactivation of METTL11
.
Ser27 is located in a highly conserved sequence in the N-terminal region of METTL1, and it is interesting to note that structural information
about this region is missing in the crystal structure of all resolved METTL1 or homologous proteins.
Through mutation (or truncation) experiments, the authors demonstrated that the conserved region of the N-terminus is necessary for METTL1 to function normally, but the role of the N-terminus and the working mechanism by which phosphorylation modifications regulate its activity are unclear
.

In order to obtain the overall impression of the N-terminal, the research team used AlphaFold2 to predict
the full-length METTL1-WDR4 complex.
The prediction results show that the N-terminal of METTL1 uses U-shaped conformation to shuttle through the active pocket, while the N-terminal conserved region participates in the construction of the METTL1 active pocket, which is very consistent
with the experimental results of electron microscopy and NMR.
Ser27 is located in the center of the active pocket but does not interact
with important amino acid residue formation.
Further serial mutant experiments found (Ser27 mutations to A, C, I, D, K, W, etc.
) that Ser27 reduced METTL1 activity when mutated to longer side chain amino acids, while shorter side chains did not
.
Therefore, the authors proposed the hypothesis that METTL1 inhibits the activity of methyltransferase by steric hindrance induced by Ser27 phosphorylation, and verified the hypothesis by experiments such as NMR titration
.

Figure 3.
Model of human METTL1-WDR4 operation and activity regulation

In a research paper titled Structures and mechanisms of tRNA methylation by METTL1–WDR4, published by Yunsun Nam's team at UT southwestern Medical Center at the same time, the researchers used crystallography and cryo-EM to resolve METTL1, METTL1-WDR4 and METTL1-WDR4-tRNA and other structures, and the catalytic activity center was discussed in detail, revealing that in addition to the N terminal of METTL1, the C terminal of WDR4 is also very important
for the normal activity of METTL1.

The overall conclusion of the two papers is consistent and complementary, and the working mechanism of METTL1 and WDR4-modified tRNA is more comprehensively explored
.
Therefore, this paper lays an important foundation
for the development of novel therapeutics for understanding the pathogenesis of METTL1-WDR4-mediated m7G modification-related cancers and other diseases, and for the development of m7G-modified inhibitors or drugs to provide important structural and mechanistic information of METTL1-WDR4 targets.

Link to the paper: class="vsbcontent_end" style="text-align: right;">(Editor: Fu Xiaoge)