Characterizing the RNA-binding activity of MEX-5

Research strategy - method specificity Target 1: how the primary sequence of MEX - 5 determines its RNA binding specificity. The CCCH-type TZF protein recognizes their RNA target (reference Hudson, Teproba) by an H-binding template between the amide group of the protein backbone and the carbonyl group and the Watson-Crick end of the base. This observation suggests that in the CCCH-type TZF protein, any change in the primary sequence leading to a change in bone structure changes the pattern of interaction between the protein and the RNA molecule, thus altering the ability of the RNA to bind to a particular sequence .

The duplex of crRNA and tracrRNA serves as a guide RNA for introducing specific localized genetic modifications based on RNA 5 'upstream of crRNA. Cas 9 binds to trac rRNA and requires a DNA binding sequence (5 'NGG 3') called prototype interval adjacent primer (PAM). After binding, Cas9 introduces DNA double strand breaks and is then genetically modified by homologous recombination (HDR) or heterologous terminal binding (NHEJ). In somatic gene therapy (SCGT), therapeutic genes are introduced into any cell other than gametes, germ cells, gametophytes or undifferentiated stem cells. Such modification affects only individual patients and not to descendants. Somatic gene therapy represents the underlying basic research and clinical research where therapeutic DNA (integrated into the genome or as an external episome or plasmid) is used to treat the disease.

Transcription is the process of using DNA genetic information to generate complementary RNA strands. The RNA strand is then processed to produce messenger RNA (mRNA), which can freely move by the cell. The mRNA molecule binds to a protein-RNA complex called ribosome located in the cytosol, where they are translated into polypeptide sequences. Ribosomes mediate the formation of polypeptide sequences based on mRNA sequences. The mRNA sequence is directly related to the polypeptide sequence by binding to the transfer RNA (tRNA) adapter molecule within the binding pocket of the ribosome. The new polypeptide is then folded into a functional three-dimensional protein molecule.

Generally, transcription begins when RNA polymerase binds to a so-called promoter sequence on a DNA molecule. In most cases, this sequence is located upstream of the transcription start site (5 'end of DNA), but it may be located downstream (3' end of mRNA). In recent years researchers have found that other DNA sequences called enhancer sequences also play an important role in transcription by providing binding sites for regulatory proteins that affect RNA polymerase activity. Binding of a regulatory protein to an enhancer sequence results in a change in chromatin structure that promotes or inhibits binding of RNA polymerase and transcription factor. The more open chromatin structure is associated with the transcription of active genes. In contrast, the more compact chromatin structure is associated with transcriptional inactivation (Figure 2).