Review

Capping of eucaryotic mRNAs

The successful use of mRNA vaccines during the Covid-19 pandemic has created a boom in mRNA therapeutic research and development. The efficacy of mRNA vaccines and therapies relies on the quality of the synthesized molecules – a key feature of which is the 5′-end cap modification. The development of analytical methods for assessing mRNA quality needs to be prioritized to enable manufacturing development, process control, and rapid assessment of batch quality before release. In this review, we provide an overview of the latest techniques in the analysis of mRNA 5′ capping. We also discuss future possibilities and challenges in quality control of mRNA products at scale.

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5′ cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here, we determine high-resolution cryoelectron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

In the last 50 years, over 150 various chemical modifications on RNA molecules, including mRNAs, rRNAs, tRNAs, and other noncoding RNAs (ncRNAs), have been identified and characterized. These RNA modifications regulate RNA biogenesis and biological functions and are widely involved in various physiological processes and diseases, including cancer. In recent decades, broad interest has arisen in the epigenetic modification of ncRNAs due to the increased knowledge of the critical roles of ncRNAs in cancer. In this review, we summarize the various modifications of ncRNAs and highlight their roles in cancer initiation and progression. In particular, we discuss the potential of RNA modifications as novel biomarkers and therapeutic targets in cancer.

Co-transcriptional capping of the nascent pre-mRNA 5′ end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5′-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

N⁷-methylguanosine (m⁷G) modification, routinely occurring at mRNA 5′ cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m⁷G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m⁷G modification remains elusive. Here, we report that mRNA internal m⁷G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m⁷G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m⁷G-modified and QKI-bound mRNA targets with a conserved “GANGAN (N = A/C/U/G)” motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m⁷G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m⁷G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Viral RNAs (vRNAs) are decorated by post-transcriptional modifications, including methylation of nucleotides. Methylations regulate biological functions linked to the sequence, structure, and protein interactome of RNA. Several RNA viruses were found to harbor 2′-O-methylations, affecting the ribose moiety of RNA. This mark was initially shown to target the first and second nucleotides of the 5′-end cap structure of mRNA. More recently, nucleotides within vRNA were also reported to carry 2′-O-methylations. The consequences of such methylations are still puzzling since they were associated with both proviral and antiviral effects. Here, we focus on the mechanisms governing vRNA 2′-O-methylation and we explore the possible roles of this epitranscriptomic modification for viral replication.