The Structure of an RNAi Polymerase Links RNA Silencing and Transcription

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-Å resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world

Insights into the pre-initiation events of bacteriophage phi6 RNA-dependent RNA polymerase : towards the assembly of a productive binary complex

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.Peer reviewe

A nationwide real-world study on dynamic ustekinumab dosing and concomitant medication use among Crohn's disease patients in Finland

Background Real-world evidence to support optimal ustekinumab dosing for refractory Crohn's disease (CD) patients remains limited. Data from a retrospective nationwide chart review study was utilized to explore ustekinumab dosing dynamics and optimization, identify possible clinical predictors of dose intensification, and to evaluate ustekinumab trough concentrations (TCs) and concomitant medication use in Finland. Methods Information gathered from17 Finnish hospitals included clinical chart data from 155 adult CD patients who received intravenous ustekinumab induction during 2017-2018. Data on ustekinumab dosing and TCs, concomitant corticosteroid and immunosuppressant use, and antiustekinumab antibodies were analyzed in a two-year follow-up, subject to availability. Results Among 140 patients onustekinumab maintenance therapy, dose optimization was required in 55(39%) of the patients, and 41/47 dose-intensified patients (87%) persisted on ustekinumab. At baseline, dose-intensified patient group had significantly higher C-reactive protein (CRP) levels, and at week 16, significantly lower ustekinumab TCs than in patients without dose intensification. Irrespective of dose optimization, a statistically significant reduction in the use of corticosteroids was observed at both 16 weeks and one year, coupled with an increased proportion of patients on ustekinumab monotherapy. Antiustekinumab antibodies were undetectable in all 28 samples from 25 patients collected throughout the study period. Conclusions Nearly a third of all CD patients on ustekinumab maintenance therapy, with a history of treatment-refractory and long-standing disease, required dose intensification. These patients persisted on ustekinumab and had significant reduction of corticosteroid use. Increased baseline CRP was identified as the sole indicator of dose intensification.Peer reviewe

The Structure of an RNAi Polymerase Links RNA Silencing and Transcription

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-Å resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world

A nationwide real-world study on dynamic ustekinumab dosing and concomitant medication use among Crohn's disease patients in Finland

Background Real-world evidence to support optimal ustekinumab dosing for refractory Crohn's disease (CD) patients remains limited. Data from a retrospective nationwide chart review study was utilized to explore ustekinumab dosing dynamics and optimization, identify possible clinical predictors of dose intensification, and to evaluate ustekinumab trough concentrations (TCs) and concomitant medication use in Finland.Methods Information gathered from17 Finnish hospitals included clinical chart data from 155 adult CD patients who received intravenous ustekinumab induction during 2017-2018. Data on ustekinumab dosing and TCs, concomitant corticosteroid and immunosuppressant use, and antiustekinumab antibodies were analyzed in a two-year follow-up, subject to availability.Results  Among 140 patients onustekinumab maintenance therapy, dose optimization was required in 55(39%) of the patients, and 41/47 dose-intensified patients (87%) persisted on ustekinumab. At baseline, dose-intensified patient group had significantly higher C-reactive protein (CRP) levels, and at week 16, significantly lower ustekinumab TCs than in patients without dose intensification. Irrespective of dose optimization, a statistically significant reduction in the use of corticosteroids was observed at both 16 weeks and one year, coupled with an increased proportion of patients on ustekinumab monotherapy. Antiustekinumab antibodies were undetectable in all 28 samples from 25 patients collected throughout the study period.Conclusions Nearly a third of all CD patients on ustekinumab maintenance therapy, with a history of treatment-refractory and long-standing disease, required dose intensification. These patients persisted on ustekinumab and had significant reduction of corticosteroid use. Increased baseline CRP was identified as the sole indicator of dose intensification.</div

Nontemplated Terminal Nucleotidyltransferase Activity of Double-Stranded RNA Bacteriophage φ6 RNA-Dependent RNA Polymerase ▿

The replication and transcription of double-stranded RNA (dsRNA) viruses occur within a polymerase complex particle in which the viral genome is enclosed throughout the entire life cycle of the virus. A single protein subunit in the polymerase complex is responsible for the template-dependent RNA polymerization activity. The isolated polymerase subunit of the dsRNA bacteriophage φ6 was previously shown to replicate and transcribe given RNA molecules. In this study, we show that this enzyme also catalyzes nontemplated nucleotide additions to single-stranded and double-stranded nucleic acid molecules. This terminal nucleotidyltransferase activity not only is a property of the isolated enzyme but also is detected to take place within the viral nucleocapsid. This is the first time terminal nucleotidyltransferase activity has been reported for a dsRNA virus as well as for a viral particle. The results obtained together with previous high-resolution structural data on the φ6 RNA-dependent RNA polymerase suggest a mechanism for terminal nucleotidyl addition. We propose that the activity is involved in the termination of the template-dependent RNA polymerization reaction on the linear φ6 genome

Structural explanation for the role of Mn^2+ in the activity of Φ6 RNA-dependent RNA polymerase

The biological role of manganese (MN^2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage Φ6 is one of the best understood examples of this important class of polymerases. We have probed the role of MN^2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn^2+ -binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn^2+, reduced RNA binding and a compromised elongation rate. Loss of Mn^2+ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn^2+ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn^2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization

Evolutionary Relationships

<div><p>(A) Evolutionary phylogenetic tree for known polymerases based on structural similarity (for description of the method see [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040434#pbio-0040434-b043" target="_blank">43</a>]). The “right-handed” polymerases, “double-barrel” polymerases, and polβ appear to form three separate, unrelated families. Within the “double-barrel” family, the DPBB2 domains containing the active site aspartate residues are more structurally conserved than DPBB1. Branches are coloured according to structural fold: green, right hand (dark, cellular; and light, viral), Polβ, blue; and DPBB-containing fold, dark magenta. Since we do not believe that all polymerases originate from a common ancestor, the central node of the tree is shaded grey. The key to the additional structures is: Yeast DPBB1, yeast RNApolII DPBB from Rbp2; Yeast DPBB2, yeast RNApolII DPBB from Rbp1; Bact. DPBB1, bacterial β subunit DPBB; Bact. DPBB2, bacterial β′ subunit DPBB; Polβ, rat DNA polymerase β; T7pol, bacteriophage T7 DdRP; KF, Klenow fragment of DNA polymerase I; HIV1-RT, human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT); λ3 reo, reovirus RdRP; Φ6, Φ6 bacteriophage RdRP; HCV, hepatitis C virus RdRP; BVDV, bovine viral diarrhoea virus RdRP; PV, poliovirus RdRP; RHDV, rabbit hemorrhagic disease virus RdRP; NV, Norwalk virus RdRP; HRV, human rhinovirus RdRP; and FMDV, foot–and-mouth disease virus RdRP.</p> <p>(B) Originally, in an all RNA world, RNA self-replicates until the advent of a protein-based, primeval RNA-dependent RNA polymerase. Initially this possesses a single DPBB domain on a single polypeptide chain. Gene duplication leads to a polypeptide chain containing two copies of the DPBB domain. Differentiation of the two DPBB domains then results in QDE-1–like RdRPs. Emergence of DNA and associated increase of complexity lead to segregation of the DPBB into different polypeptidic chains, giving rise to the complex multi-subunit DdRP machinery observed today.</p></div

Structure-Based Sequence Alignment of QDE-1 ΔN and DdRPs

<p>Alignment of QDE1 ΔN (top sequence), bacterial (middle sequence, orange), and yeast (bottom sequence, green) polymerases is based on structurally equivalent residues, as determined by SHP [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040434#pbio-0040434-b041" target="_blank">41</a>]. Residues structurally equivalent in all polymerases are shaded purple, green (light for Rbp1, dark for Rp2) if equivalent in yeast DdRP and QDE1 ΔN, and orange if only equivalent in QDE1 ΔN and bacterial DdRP. Invariant residues are shaded in red. Conserved sequence motifs identified in cRdRPs are represented as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040434#pbio-0040434-sg001" target="_blank">Figure S1</a>, marked on QDE1 sequence. QDE1 secondary structure elements are shown on top, coloured according to domain definition (slab, blue; catalytic, deep purple; neck, pink; and head, red). DPBB1 and DPBB2 are outlined by deep purple boxes. The flap sub-domain and the potential “bridge helix” are also represented by boxes, coloured light purple and grey, respectively.</p