Chongfan Technology
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30
2024
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08
In 2024, Feng Zhang's team cooperated to issue 6 research results such as Cell/Nature/Science.
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Reverse transcription is frequently used for cellular functions and has been implicated in protection against viral infection in prokaryotes, but the underlying defense mechanisms are often unknown.
On August 29, 2024, the Massachusetts Institute of Technology Zhang Feng team inSciencePublished online entitled“Phage-triggered reverse transcription assembles a toxic repetitive gene from a noncoding RNA”The research paper,The study foundPhage-triggered reverse transcription assembled a toxic duplicate gene from non-coding RNA.
This study shows that in the DRT2 defense system, reverse transcriptase binds adjacent tangle non-coding RNAs.After phage infection, the template region of this RNA is reverse transcribed into a series of tandem repeats that reconstitute the promoter and open reading frame, allowing expression of the toxic repeat protein and abortive infection response.The biochemical reconstitution of this activity provides the molecular basis for repeated synthesis. Gene synthesis from noncoding RNA is a new mode of genetic regulation in prokaryotes.
In addition, on August 28, 2024, the MIT/Broad Institute Zhang Feng team was inCellPublished online entitled“Structural insights into the diversity and DNA cleavage mechanism of Fanzor”The research paper,The studyThe structures of Fzs from three different organisms are described.The study found that regardless of the length of the ω RNA, Fzs have a common ω RNA interaction interface, which varies greatly between species.The study also revealed the DNA recognition pattern and unwinding ability of Fz and the presence of non-canonical catalytic sites. These structures demonstrate how the protein conformation of Fz moves to allow double-stranded DNA to bind to the active site within the R-loop.Mechanically, structural detection in different states suggests that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, which regulates the activation of nucleases and the displacement of the DNA duplex at the single cleavage site.
On August 6, 2024, the Zhang Feng team at the Broad InstituteMolecular CellPublished online entitled"Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system"'s research paper, which studiesdescribesSelenomonas sp.The cryo-electron microscopic structure of HNH-Cascade (SsCascade) complex with target DNA and its mechanism of action were characterized.The cascade scaffold is complemented by HNH domains, forming a circular structure in which the unwrapped target DNA is precisely cleaved. This structure visualizes a unique hybrid of two scalable biological systems-cascade, an evolutionary platform for programmable DNA effectors, and HNH nucleases, an adaptive domain with an enzyme activity profile (Click to read）。
On June 11, 2024, Harvard Medical School/MIT/Broad Institute Zhang Feng and other teams worked together inMobile DNAOnline publication entitled"Internal initiation of reverse transcription in a Penelope-like retrotransposon"The research paper,Using a protein purified from Escherichia coli, the study reported that (Anolis carolinensisThe unique in vitro properties of PLE extracted in) reveal mechanistic aspects not found in other retrotransposons.The study found that reverse transcription is initiated at two adjacent sites in the transposon RNA, which are not homologous to the DNA being cut, a feature reflected in the genome's "tail" feature, which is shared and unique among PLEs.The study, which for the first time activates PLE in vitro, provides a starting point for understanding the mobilization and biology of PLE (Click to read）。
On May 29, 2024, Osamu Nureki of the University of Tokyo and Zhang Feng of the Broad Institute worked together inNatureOnline published the title"Structural basis for pegRNA-guided reverse transcription by a prime editor"research paper, which demonstratesCryo-electron microscopic structures of the RTΔRNaseH-pegRNA-target DNA complex in various states were SpCas9-M-MLV.Termination structure as well as functional analysis showed that M-MLV RT extended reverse transcription beyond the intended site, resulting in scaffold-derived binding, causing unwanted editing at the target site. Structural comparisons of pre-initiation, initiation, and extension states indicate that the M-MLV RT maintains a consistent position relative to SpCas9 during reverse transcription, while pegRNA-synthesized DNA hybridized duplexes build up along the SpCas9 surface. On the basis of structural insights, the study rationally designed pegRNA variants and lead editing variants in which the M-MLV RT was fused in SpCas9.Overall, the results of the study provide structural insights into the stepwise mechanism of pilot editing and will pave the way for the development of a multifunctional pilot editing toolbox (Click to read）。
On March 4, 2024, the Zhang Feng team at the Broad InstitutePNASOnline publication entitled"人类副肿瘤抗原Ma2 （PNMA2） forms icosahedral capsids that can be engineered for mRNA delivery"The research paper, the studyHuman PNMA proteins were systematically explored and it was found that human cells secreted many PNMA.The study determined that pNMA2 efficiently forms icosahedral capsids, but does not naturally encapsulate nucleic acids. In this study, the cryo-electron microscopy (cryo-EM) structure of pNMA2 was analyzed, and the engineered pNMA2 (ePNMA2) particles with RNA packaging capacity were designed using this structure.Recombinantly purified ePNMA2 protein packages mRNA molecules into icosahedral capsids and can serve as a delivery vehicle for mammalian cell lines, demonstrating the potential of engineered endogenous capsids as a means of delivery for nucleic acid therapy.
RNA templated DNA synthesis or reverse transcription is a fundamental way to reverse the flow of genetic information. This activity is usually associated with the mobility of RNA-based mobile genetic elements (such as retroviruses, retrotransposons, and group II introns) catalyzed by reverse transcriptase (RT), but has also been used for cellular functions many times.In eukaryotes, the RT activity of telomerase maintains genome integrity, and the RT domain protein forms the core of the splice body, thereby removing introns in the precursor. In prokaryotes, RT is often associated with phage defense.For example, RT can mediate the acquisition of CRISPR spacers from RNA, while retron RT synthesizes specialized RNA/DNA/protein complexes that function through a variety of emerging mechanisms to limit phage proliferation.
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