Modified nucleotides on RNAs have been investigated for over six decades for their potential role in regulating gene expression and protein synthesis across a wide range of organisms, from animals to plants and fungi, as well as in viral genetic materials. Among them, mRNA methylation stands out with its dynamic nature, which underscores the adaptability of the epitranscriptome in developmental transitions and response to environmental stress, especially in plants. Advances in next-generation sequencing methods have revealed the specific sequence contexts of mRNA methylation, uncovering their involvement in gene regulatory networks. Additionally, genetic perturbations on the writers, erasers, and readers of m6A and m5C expanded our understanding of the physiological function and the mode of action of these modifications. In this review, we highlight recent advances in understanding how mRNA fate decisions, mainly determined by m6A and m5C RNA methylation, shape stress response and development in plants.

Eukaryotes also employ three different polymerases that each transcribe a different subset of genes. Eukaryotic mRNAs are usually monogenic, meaning that they specify a single protein. Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase. The features of eukaryotic mRNA synthesis are markedly more complex those of prokaryotes. Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more. Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template. RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes (Table 15.1). The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation.

produce different types of RNA, including: Messenger RNA (mRNA) An RNA copy of the coding strand of a gene which is translated into a polypeptide chain In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template strand. Along with the enzyme helicase, RNAP locally opens the double-stranded DNA so that one strand of the exposed nucleotides can be used as a template for the synthesis of Messenger RNA (mRNA)—template for the synthesis of proteins by ribosomes. Non-coding RNA or "RNA genes"—a broad class of genes that encode RNA that is not translated into protein. The most prominent examples of RNA genes are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation. However, since the late 1990s, many new RNA genes have been found, and thus RNA genes may play a much more significant role than previously thought. Transfer RNA (tRNA)—transfers specific amino acids to growing polypeptide chains at the ribosomal site of protein synthesis during translation Ribosomal RNA (rRNA)—a component of ribosomes Micro RNA—regulates gene activity Catalytic RNA (Ribozyme)—enzymatically active RNA molecules RNAP accomplishes de novo synthesis. It is able to do this because specific interactions with the initiating nucleotide hold RNAP rigidly in place, facilitating chemical attack on the incoming nucleotide. Such specific interactions explain why RNAP prefers to start transcripts with ATP (followed by GTP, UTP, and then CTP). In contrast to DNA polymerase, RNAP includes helicase activity, therefore no separate enzyme is needed to unwind DNA.

Cell-free translation system using phosphorothioate-containing mRNA. Phosphorothioate-containing RNAs were generated by transcription of template DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) (NTP alpha S) and T7 RNA polymerase. The substitution of mRNA by phosphorothioate increased the efficiency of protein synthesis by stabilizing the mRNAs in prokaryotic cell-free translation systems. The substituted mRNAs were also shown to be applicable to the continuous cell-free translation system developed by Spirin and coworkers. Published in Nucleic acids symposium series (1991)

Changes in template activity of protein and globin mRNA during Chironomus development. A wheat-germ cell-free protein synthesizing system was established that efficiently translates fourth instar mRNA from Chironomus thummi. The translation products were analyzed by double immunoprecipitation with specific antibodies against Chironomus hemoglobins. The predominant translation products were shown to be globins, comprising about 50% of total protein synthesized. Two globins, globins 2 and 3, which are specific for the fourth instar in vivo, constitute most of the globin produced. The wheatgerm system translates also efficiently total cytoplasmic RNA, purified from animals at successive developmental stages. The kinetics of protein synthesis during development indicate that Chironomus mRNA template activity is low during larval molting and at metamorphosis. RNA from intermolt larvae generally demonstrates high template activity. In the fourth instar two distinct peaks of activity are resolved, one associated with newly molted fourth instars and a second one associated with the prepupal stage. These RNAs direct the synthesis mostly of non-globin proteins.

SEVERAL steps in the replication of the small phage Qβ can be reconstructed under cell-free conditions. Net synthesis of biologically active RNA “plus” strands has been achieved in vitro using RNA from virus particles as a template and RNA dependent RNA polymerase or “replicase” from infected E. coli cells1. This reaction proceeded through complementary RNA or “minus” strands as intermediate templates2. The plus strand-dependent reaction required a “host factor” HF I in addition to purified replicase3–6,7. Furthermore, three of four phage coded proteins can be synthesized in vitro using Qβ RNA as an mRNA and an extract from uninfected E. coli as a protein synthesizing system8,9. Coat protein and the phage specific subunit of replicase (R protein) when synthesized in vitro became bound to Qβ RNA8,10 as would be expected for the authentic proteins. Cell-free synthesis of enzymatically active Qβ replicase, however, has not been reported so far.

by miRNAs (microRNAs). These RNA strands can cleave mRNA strands they are complementary to and will thus stop translation. Translation can also be regulated Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism. The steps of protein synthesis include transcription, translation, and post translational modifications. During transcription, RNA polymerase transcribes a coding region of the DNA in a cell producing a sequence of RNA, spe Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism. The steps of protein synthesis include transcription, translation, and post translational modifications. During transcription, RNA polymerase transcribes a coding region of the DNA in a cell producing a sequence of RNA, specifically messenger RNA (mRNA). This mRNA sequence contains codons: 3 nucleotide long segments that code for a specific amino acid. Ribosomes translate the codons to their respective amino acids. In humans, non-essential amino acids are synthesized from intermediates in major metabolic pathways such as the Citric Acid Cycle. Essential amino acids must be consumed and are made in other organisms. The amino acids are joined by peptide bonds making a polypeptide chain. This polypeptide chain then goes through post translational modifications and is sometimes joined with other polypeptide…

Translational requirement for La Crosse virus S-mRNA synthesis: a possible mechanism. Ongoing protein synthesis is required for La Crosse S-mRNA synthesis in vivo, and complete S-mRNA can be made in vitro only in the presence of an active rabbit reticulocyte lysate. Using in vitro systems based on the polymerase activity of purified virions, we further support the notion that it is translation of the nascent mRNA that is required for complete transcription. Since replacement of guanosine with inosine in the nascent mRNA substitutes for the translational requirement, it appears that translation is required to prevent interactions of the nascent chain from taking place, which, if not prevented, lead to premature termination. These interactions appear to be between the nascent mRNA chain and its nucleocapsid template. A model for the translational requirement for complete S-mRNA synthesis is presented. Published in Journal of virology (1987)

Protein synthesis in cell-free protein synthesis systems often exhibits nonintuitive input-output relationships. In the PURE system, a reconstituted cell-free system, protein production peaked at low elongation factor Tu (EF-Tu) concentrations and decreased at higher concentrations, resulting in a characteristic bell-shaped profile. Here, we investigated the origin of this behavior using a detailed mechanistic model of translation in the PURE system, designated as ePURE, which describes the reaction dynamics of hundreds of molecular species and reactions. Our computational analysis suggested that excess EF-Tu sequesters the initiator tRNA (tRNA^fMet) into nonproductive EF-Tu·GTP·Met-tRNA^fMet complexes, thereby depleting the pool of initiator tRNA available for translation initiation. This suppression arises from competition for a limited molecular resource rather than from direct inhibition. Based on this mechanism, we predicted that increasing the concentrations of tRNA^fMet and methionyl-tRNA formyltransferase would eliminate the bell-shaped dependence, and experimentally confirmed this prediction. Under these modified conditions, the bell-shaped response disappeared and protein production was enhanced. These findings demonstrate how mechanistic computational models can reveal hidden constraints underlying nonintuitive input-output relationships in complex biochemical networks and guide the rational optimization of cell-free protein synthesis systems.