Weislow, I. S. | bioRxiv Science

bioRxiv2026

Direct RNA Sequencing Reveals Stress-Dependent and Pathway-Specific rRNA Modification Reprogramming During 50S Biogenesis

Ribosomal RNA (rRNA) modification and processing are essential steps in ribosome assembly. Using Oxford Nanopore direct RNA sequencing, we simultaneously detect and quantify eight classes of 23S rRNA modifications in the mature 50S large subunit (LSU) from Escherichia coli cells expressing either wild-type DbpA or the helicase-inactive R331A DbpA variant, as well as in two LSU assembly intermediates, 35S and 45S, which accumulate along distinct maturation pathways in R331A DbpA expressing cells. In addition, we analyze 3'-end processing of 23S and 5S rRNAs across these particles. Many 23S rRNA modifications are incorporated at similar levels in LSU assembly intermediates and mature 50S subunits from both wild-type and R331A DbpA expressing cells, indicating that these modifications are incorporated prior to intermediate accumulation and are not preferentially reprogrammed under R331A DbpA induced assembly stress. In contrast, a subset of three modifications exhibits altered incorporation patterns. N2-methyladenosine 2507 incorporation is reduced in the 50S LSU from R331A DbpA expressing cells compared with the cells expressing wild-type DbpA, whereas pseudouridine ({Psi}) 2508 is increased. In addition, {Psi} 2608 is reduced in the 50S subunit from R331A DbpA expressing cells compared with the 35S and 45S intermediates from the same cells and the 50S subunit from wild-type cells. Because the 35S and 45S pathways account for only [~]40% of ribosome assembly in R331A DbpA expressing cells, these findings demonstrate that {Psi}2608 incorporation is selectively reprogrammed across alternative in vivo assembly routes, revealing an additional regulatory layer in ribosome biogenesis.

biochemistry↗

bioRxiv2024

Substrate Specificities of DDX1: A Human DEAD-box protein

DDX1 is a human protein which belongs to the DEAD-box protein family of enzymes and is involved in various stages of RNA metabolism from transcription to decay. Many members of the DEAD-box family of enzymes use the energy of ATP binding and hydrolysis to perform their cellular functions. On the other hand, a few members of the DEAD-box family of enzymes bind and/or hydrolyze other nucleotides in addition to ATP. Furthermore, the ATPase activity of DEAD-box family members is stimulated differently by nucleic acids of various structures. The identity of the nucleotides that the DDX1 hydrolyzes and the structure of the nucleic acids upon which it acts in the cell remain largely unknown. Identifying the DDX1 proteins in vitro substrates is important for deciphering the molecular roles of DDX1 in cells. Here we identify the nucleic acid sequences and structures supporting the nucleotide hydrolysis activity of DDX1 and its nucleotide specificity. Our data demonstrate that the DDX1 protein hydrolyzes only ATP and deoxy-ATP in the presence of RNA. The ATP hydrolysis activity of DDX1 is stimulated by multiple molecules: single-stranded RNA molecules as short as ten nucleotides, a blunt-ended double-stranded RNA molecule, a hybrid of a double-stranded DNA-RNA molecule, and a single-stranded DNA molecule. Under our experimental conditions, the single-stranded DNA molecule stimulates the ATPase activity of DDX1 at a significantly reduced extent when compared to the other investigated RNA constructs or the hybrid double-stranded DNA/RNA molecule.

biochemistry↗