Branched chemically modified poly(A) tails enhance the translation capacity of mRNA.

Although messenger RNA (mRNA) has proved effective as a vaccine, its potential as a general therapeutic modality is limited by its instability and low translation capacity. To increase the duration and level of protein expression from mRNA, we designed and synthesized topologically and chemically modified mRNAs with multiple synthetic poly(A) tails. Here we demonstrate that the optimized multitailed mRNA yielded ~4.7-19.5-fold higher luminescence signals than the control mRNA from 24 to 72 h post transfection in cellulo and 14 days detectable signal versus <7 days signal from the control in vivo. We further achieve efficient multiplexed genome editing of the clinically relevant genes Pcsk9 and Angptl3 in mouse liver at a minimal mRNA dosage. Taken together, these results provide a generalizable approach to synthesize capped branched mRNA with markedly enhanced translation capacity.

PPM1H is down-regulated by ATF6 and dephosphorylates p-RPS6KB1 to inhibit progression of hepatocellular carcinoma.

We have shown previously that polymorphism of activating transcription factor 6 (ATF6) is associated with susceptibility to hepatocellular carcinoma (HCC). Therefore, genes down-regulated by ATF6 might play a tumor-suppressing role. In the present study, we identified that expression of protein phosphatase magnesium- or manganous-dependent 1H (PPM1H) mRNA and protein can be inhibited by ATF6 in hepatoma cells and mice with liver Atf6 knockdown. Tumor tissues from 134 HCC patients were analyzed by immunohistochemistry, and PPM1H exhibited higher expression levels in adjacent para-cancer tissues than in HCC tissues. Therefore, patients with higher expression of PPM1H had a better prognosis. PPM1H inhibited proliferation, migration, and invasion of hepatoma cells. In addition, PPM1H inhibited induced HCC nodule formation as well as tumor xenograft growth in diethylnitrosamine/CCl4-induced HCC mouse model and nude mouse tumorigenicity assay, respectively. A 3D model of PPM1H was obtained by homology multi-template modeling, and ribosomal protein S6 kinase B1 (RPS6KB1) in the bone morphogenetic protein (BMP)/transforming growth factor ß (TGF-ß) pathway was screened out as the potential substrate of PPM1H by Rosetta. PPM1H could directly dephosphorylate p-RPS6KB1. To conclude, we discovered RPS6KB1 as a new PPM1H dephosphorylation substrate. PPM1H exhibited a suppressive effect on HCC progression by dephosphorylating p-RPS6KB1.

Photocatalytic Chemical Crosslinking for Profiling RNA-Protein Interactions in Living Cells.

The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA-protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5' untranslated regions of SARS-CoV-2 RNA.

Chemically Modified mocRNAs for Highly Efficient Protein Expression in Mammalian Cells.

mRNA has recently been established as a new class of therapeutics, due to its programmability and ability to produce proteins of interest rapidly in vivo. Despite its demonstrated utility, mRNA as a protein expression platform remains limited by its translational capacity and RNA stability. Here, we introduce messenger-oligonucleotide conjugated RNAs (mocRNAs) to enable site-specific, robust, and modularized encoding of chemical modifications for highly efficient and stable protein expression. In mocRNA constructs, chemically synthesized oligonucleotides are ligated to the 3' terminus of mRNA substrates to protect poly(A) tails from degradation, without compromising their potency in stimulating translation. As a proof-of-concept, mocRNAs modified by deadenylase-resistant oligonucleotides result in augmented protein production by factors of 2-4 in human HeLa cells and by 10-fold in primary rat cortical neuronal cultures. By directly linking enzymatic and organic synthesis of mRNA, we envision that the mocRNA design will open new avenues to expand the chemical space and translational capacity of RNA-based vectors in basic research and therapeutic applications.