Noncanonical metabolite RNA caps: Classification, quantification, (de)capping, and function.

The 5' cap of eukaryotic mRNA is a hallmark for cellular functions from mRNA stability to translation. However, the discovery of novel 5'-terminal RNA caps derived from cellular metabolites has challenged this long-standing singularity in both eukaryotes and prokaryotes. Reminiscent of the 7-methylguanosine (m7G) cap structure, these noncanonical caps originate from abundant coenzymes such as NAD, FAD, or CoA and from metabolites like dinucleoside polyphosphates (NpnN). As of now, the significance of noncanonical RNA caps is elusive: they differ for individual transcripts, occur in distinct types of RNA, and change in response to environmental stimuli. A thorough comparison of their prevalence, quantity, and characteristics is indispensable to define the distinct classes of metabolite-capped RNAs. This is achieved by a structured analysis of all present studies covering functional, quantitative, and sequencing data which help to uncover their biological impact. The biosynthetic strategies of noncanonical RNA capping and the elaborate decapping machinery reveal the regulation and turnover of metabolite-capped RNAs. With noncanonical capping being a universal and ancient phenomenon, organisms have developed diverging strategies to adapt metabolite-derived caps to their metabolic needs, but ultimately to establish noncanonical RNA caps as another intriguing layer of RNA regulation. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability.

Chemoenzymatic strategies for RNA modification and labeling.

RNA is a central molecule in numerous cellular processes, including transcription, translation, and regulation of gene expression. To reveal the numerous facets of RNA function and metabolism in cells, labeling has become indispensable and enables the visualization, isolation, characterization, and even quantification of certain RNA species. In this review, we will cover chemoenzymatic approaches for covalent RNA labeling. These approaches rely on an enzymatic step to introduce an RNA modification before conjugation with a label for detection or isolation. We start with in vitro manipulation of RNA, sorted according to the enzymatic reaction exploited. Then, metabolic approaches for co- and post-transcriptional RNA labeling will be treated. We focus on recent advances in the field and highlight the most relevant applications for cellular imaging, RNA isolation and sequencing.

Cryptic Production of trans-3-Hydroxyproline in Echinocandin B Biosynthesis.

Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: trans-4-hydroxy-l-proline and, in most echinocandin structures, (trans-2,3)-3-hydroxy-(trans-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by Glarea lozoyensis, both amino acids are found in pneumocandin A0, while in pneumocandin B0 the latter residue is replaced by trans-3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by Aspergillus species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Aspergillus pachycristatus Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by CryptosporiopsisIMPORTANCE Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A0 and B0 are produced by the fungus Glarea lozoyensis They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A0 and B0, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in Aspergillus pachycristatus produces the same hydroxyprolines, with an increased proportion of trans-3-hydroxyproline. However, echinocandin B biosynthesis does not require trans-3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in G. lozoyensis and A. pachycristatus provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.

Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis-Selective Bacterial Proline Hydroxylases that Discriminates l-Proline.

Proline hydroxylases are iron(II)/2-oxoglutarate-dependent enzymes that hydroxylate l-proline and derivatives, such as lpipecolic acid, which is the six-membered-ring homologue of l-proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l-pipecolic acid and trans-3- and trans-4-methyl-l-proline, but virtually no l-proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l-proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l-proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site-directed mutagenesis in GetF did not yield variants that accepted l-proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l-proline relative to that of preferred substrates.