Liang Zhang

In recent years, along with many advances in cancer immunotherapy, immune checkpoints, a class of immunomodulatory proteins involved in the negative regulation of the immune system, have attracted increasing attention.

Chromatin contains not only heterochromatin (HC) and euchromatins (EC) but also facultative heterochromatin (fHC), which experience the dynamic remodeling between HCs and ECs by different regulators. The regulation of fHCs involves lots of different cell functions, like genomic stability and gene transcription. Heterochromatin protein 1 (HP1) recognizes methylated H3K9 and reshapes the chromatin into the fHCs through liquid–liquid phase separation (LLPS). Among the three members of the HP1 family, HP1α can condensate by itself and HP1β forms granules with the help of TRIM28, while the HP1γ cannot phase separation alone either and the coordinator is still unclear. So, in this study, we investigated the molecular mechanism of how HP1γ interacts with TRIM66 through PxVxL motif. Based on that, we examined the key regions that controlled the TRIM66-HP1γ co-phase separation behaviors both in vitro and in vivo. Furthermore, we proved that the liquid granules of TRIM66-HP1γ and chromatin highly correlated with H3K9me3 sites, which indicated the relationship with DNA damage response. Finally, combined with our previous study, we proposed the system for how TRIM66 remodeled the chromatin into compressed fHC through the TRIM66-HP1γ-H3K9me3 axis with liquid–liquid phase separation.

Telomeres are the complexes composed of repetitive DNA sequences and associated proteins located at the end of chromatin. As a result of the DNA replication ending issue, telomeric DNA shortens during each cell cycle. The shelterin protein complex caps telomeric ends and forms a high-order protein–DNA structure to protect telomeric DNA. The stability of telomeres is critical for cellular function and related to the progression of many human diseases. Telomeric repeat-containing RNA (TERRA) is a noncoding RNA transcribed from telomeric DNA regions. TERRA plays an essential role in regulating and maintaining the stability of telomeres. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins associated with complex and diverse biological processes. hnRNPA1 can recognize both TERRA and telomeric DNA. Previous research reported that hnRNPA1, TERRA, and POT1, a component of the shelterin complex, work coordinately and displace replication protein A from telomeric single-stranded DNA after DNA replication, promoting telomere capping to preserve genomic integrity. However, the detailed molecular mechanism has remained unclear for >20 years. Here, our study revealed the molecular structure through which the hnRNPA1 UP1 domain interacts with TERRA and identified critical residues on the interacting surface between UP1 and TERRA. Furthermore, we proved that nucleic acids significantly increase the phase-separating ability of hnRNPA1, while disrupting the UP1–TERRA interaction extraordinarily affects hnRNPA1 droplet formation both in vitro and in vivo. Taken together, these data reveal the molecular mechanism of the phase separation of hnRNPA1 and TERRA and the potential contribution of the droplets to maintaining genomic stability.

Immunotherapy, including immune checkpoint inhibitors and adoptive cell transfer, has obtained great progress, but their efficiencies vary among patients due to the genetic and epigenetic differences. Human MEX3B (hMEX3B) protein is an RNA-binding protein that contains two KH domains at the N-terminus and a RING domain at its C-terminus, which has the activity of E3 ubiquitin ligase and is essential for RNA degradation. Current evidence suggests that hMEX3B is involved in many important biological processes, including tumor immune evasion and HLA-A regulation, but the sequence of substrate RNA recognized by hMEX3B and the functional molecular mechanisms are unclear. Here, we first screened the optimized hMEX3B binding sequence on the HLA-A mRNA and reported that the two tandem KH domains can bind with their substrate one hundred times more than the individual KH domains. We systematically investigated the binding characteristics between the two KH domains and their RNA substrates by nuclear magnetic resonance (NMR). Based on this information and the small-angle X-ray scattering (SAXS) data, we used molecular dynamics simulations to obtain structural models of KH domains in complex with their corresponding RNAs. By analyzing the models, we noticed that on the KH domains’ variable loops, there were two pairs of threonines and arginines that can disrupt the recognition of the RNA completely, and this influence had also been verified both in vitro and in vivo. Finally, we presented a functional model of the hMEX3B protein, which indicated that hMEX3B regulated the degradation of its substrate mRNAs in many biological processes. Taken together, our research illustrated how the hMEX3B protein played a key role in translation inhibition during the immune response to tumor cells and provided an idea and a lead for the study of the molecular mechanism and function of other MEX3 family proteins.

Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The m⁴C methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing m⁴C839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli. Here, we reported a series of crystal structures of human METTL15–hsRBFA–h44–SAM analog, METTL15–hsRBFA–SAM, METTL15–SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.

Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency ‘stochastic’ byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.