RNA polymerases usually use a DNA or RNA template to direct synthesis. Other enzymes add nucleotides to the end of an existing RNA molecule. Could a protein instead assemble an RNA chain directly from free nucleotides, without either a template or a pre-existing RNA strand?
To investigate this question, the researchers searched predicted plant protein structures for catalytic cores resembling that of human cGAS, an enzyme that joins nucleotides to make a cyclic signaling molecule. The search led them to OsNTP4, a rice protein with conserved acidic residues near its predicted active site. Structural similarity suggested a place to look, but did not establish what reaction the plant protein might perform.
Experiments with purified OsNTP4 provided the answer. When supplied with free nucleoside triphosphates, the protein generated products of varying lengths without an added nucleic-acid template or primer. Products made separately from UTP, ATP and CTP were sensitive to RNase treatment, and nuclear magnetic resonance analysis identified the canonical 3′–5′ phosphodiester linkages of RNA in these products. Mutating any one of three conserved acidic residues abolished the activity, and the reaction required divalent metal ions.
The team also examined whether the activity could arise from material carried through protein purification. Intact-protein mass spectrometry, tests for associated nucleotides, and activity assays using extensively washed OsNTP4 crystals provided independent evidence that the observed reaction is intrinsic to OsNTP4.
The finding extends beyond one protein. OsNTP10, another rice protein, and AtURT1, an Arabidopsis enzyme previously studied for adding uridines to existing RNA ends, also generated RNA products under conditions without an added template or primer. These results indicate that related plant nucleotidyltransferases can support both RNA-end extension and direct synthesis from free nucleotide substrates.
A preliminary sequencing analysis further showed that OsNTP4 produces a heterogeneous RNA population with broad distributions of length, base-composition, and homopolymer. The biological function of direct RNA synthesis by plant NTP proteins remains open. For now, the study establishes a previously unrecognized biochemical capability of these plant proteins. It also provides a starting point for discovering how the reaction works and whether plants use it to make RNA in vivo.
DOI：10.15302/vita.2026.10.0078