Search

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured “superfolder” mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

Background The DNA non-homologous end joining repair gene XRCC7 is one of the most important genes in the DNA double-strand break (DSBs) repair. It is supposed that DNA repair gene malfunction is the main risk factor in various malignancies. The XRCC7 G6721T (rs7003908) polymorphism impact was investigated on the splicing regulation that cause mRNA instability. The goal of the present hospital-based study was to investigate the association between the common genetic polymorphism of XRCC7 G6721T (rs7003908) and risk of acute lymphoblastic leukemia (ALL). This hospital-based case–control study was performed on 99 ALL patients versus 200 healthy children, as the control group, which were frequent matched by age with cases. The polymorphism of XRCC7 was determined using an RFLP-PCR technique. Results The GT (OR = 1.485, 95% CI 0.765–2.334, P = 0.243) and TT (OR = 1.655, 95% CI 00.875–3.128, P = 0.121) genotypes had no significant effect on the risk of ALL, in comparison with the GG genotype. However, TT genotype (OR = 1.996, 95% CI 1.033–3.858, P = 0.04) after adjusting for the parents’ smoking pattern showed a significant impact. Conclusions These findings suggest that the TT genotype may increase the ALL susceptibility in children when facing with a tobacco smoke.

Leaked documents show that some early commercial batches of Pfizer-BioNTech’s covid-19 vaccine had lower than expected levels of intact mRNA, prompting wider questions about how to assess this novel vaccine platform, writes Serena Tinari

Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post-translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modern high-expression transgenic Bt plants possible, with an emphasis on the reasons for the low-level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.

The labile SAUR transcripts from higher plants contain a conserved DST sequence in their 3′-untranslated regions. Two copies of a DST sequence from soybean are sufficient to destabilize reporter transcripts in cultured tobacco cells whereas variants bearing mutations in the conserved ATAGAT or GTA regions are inactive. To investigate the potential for conserved recognition components in mammalian and plant cells, we examined the function of this instability determinant in mouse NIH3T3 fibroblasts and tobacco BY2 cells. In fibroblasts, a tetrameric DST element from soybean accelerated deadenylation and decay of a reporter transcript. However, a version mutated in the ATAGAT region was equally effective in this regard, and a tetrameric DST element from Arabidopsis was inactive. In contrast, the soybean DST element was more active as an mRNA instability element than the mutant version and the Arabidopsiselement, when tested as tetramers in tobacco cells. Hence, the plant DST element is not recognized in animal cells with the same sequence requirements as in plant cells. Therefore, its mode of recognition appears to be plant-specific.

Photoinhibition mechanism is studied for ethylene production by a recombinant cyanobacterium. The kinetic pattern of the production is similar to that of substrate inhibition in enzyme reactions, whereas the photoinhibition is believed not to be substrate-related, but to originate from the enzyme for ethylene formation. In a proposed model, the inhibition is attributed to the process of enzyme synthesis, where light-dependent mRNA decomposition affects enzyme concentration. A mathematical formula derived accordingly for light dependence of ethylene production rate gives the same form as a well-known equation of substrate inhibition. The model also predicts ethylene production under dark condition, which has been observed experimentally.

Plants and other higher eukaryotes have the ability to recognize and target specific transcripts for rapid decay from among the majority of relatively stable mRNAs present within cells. However, little is known about the nature of unstable transcripts in plants, or the mechanisms that facilitate their rapid degradation. As a first step toward understanding how plants distinguish between unstable and stable transcripts, a novel differential screen was used to identify cDNAs for genes with unstable transcripts (GUTs), solely on the basis of the instability of their mRNAs. cDNA probes were prepared from tobacco cells that had been depleted of highly unstable mRNAs by treatment for 90 min with a transcriptional inhibitor, and from control, untreated cells. GUT clones were selected on the basis of weak hybridization to the former probe relative to the latter probe. Half-life measurements performed on the mRNAs hybridizing to eight GUT clones indicated that each was unstable, with a half-life on the order of about an hour or less. All eight of the cDNAs corresponded to new tobacco genes, and four showed sequence similarity with genes from other species, including the eukaryotic family of DNAJ homologs, a tomato wound-inducible protein, and histone H3. In addition to providing information about the types of transcripts that are inherently unstable in plants, the GUT clones should provide excellent tools for the identification of cis- and trans-acting determinants of mRNA instability.