Molecular mechanisms of the RNA polymerases in plant RNA-directed DNA methylation.

In plants, two atypical DNA-dependent RNA polymerases, RNA polymerase IV (Pol IV) and Pol V, and an RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) together produce noncoding RNAs (ncRNAs) to guide the plant-specific RNA-directed DNA methylation (RdDM). Although both Pol IV and Pol V have evolved from the canonical Pol II, they have adapted to different roles in RdDM. The mechanisms of their adaptation are key to understanding plant DNA methylation and the divergent evolution of polymerases. In this review, we summarize insights that have emerged from recent structural studies of Pol IV, Pol V, and RDR2 and discuss their structural features critical for efficient ncRNA production in RdDM.

Structure of plant RNA-DEPENDENT RNA POLYMERASE 2, an enzyme involved in small interfering RNA production.

In plants, the biogenesis of small interfering RNA (siRNA) requires a family of RNA-dependent RNA polymerases that convert single-stranded RNA (ssRNA) into double-stranded RNA (dsRNA), which is subsequently cleaved into defined lengths by Dicer endonucleases. Here, we determined the structure of maize (Zea mays) RNA-DEPENDENT RNA POLYMERASE 2 (ZmRDR2) in the closed and open conformations. The core catalytic region of ZmRDR2 possesses the canonical DNA-dependent RNA polymerase (DdRP) catalytic sites, pointing to a shared RNA production mechanism between DdRPs and plant RDR-family proteins. Apo-ZmRDR2 adopts a highly compact structure, representing an inactive closed conformation. By contrast, adding RNA induced a significant conformational change in the ZmRDR2 Head domain that opened the RNA binding tunnel, suggesting this is an active elongation conformation of ZmRDR2. Overall, our structural studies trapped both the active and inactive conformations of ZmRDR2, providing insights into the molecular mechanism of dsRNA synthesis during plant siRNA production.

TEM1 combinatorially binds to FLOWERING LOCUS T and recruits a Polycomb factor to repress the floral transition in Arabidopsis.

Arabidopsis TEMPRANILLO 1 (TEM1) is a transcriptional repressor that participates in multiple flowering pathways and negatively regulates the juvenile-to-adult transition and the flowering transition. To understand the molecular basis for the site-specific regulation of FLOWERING LOCUS T (FT) by TEM1, we determined the structures of the two plant-specific DNA-binding domains in TEM1, AP2 and B3, in complex with their target DNA sequences from the FT gene 5'-untranslated region (5'-UTR), revealing the molecular basis for TEM1 specificity for its DNA targets. In vitro binding assays revealed that the combination of the AP2 and B3 binding sites greatly enhanced the overall binding of TEM1 to the FT 5'-UTR, indicating TEM1 combinatorically recognizes the FT gene 5'-UTR. We further showed that TEM1 recruits the Polycomb repressive complex 2 (PRC2) to the FT 5'-UTR. The simultaneous binding of the TEM1 AP2 and B3 domains to FT is necessary for deposition of H3K27me3 at the FT 5'-UTR and for the flowering repressor function of TEM1. Overall, our data suggest that the combinatorial recognition of FT 5'-UTR by TEM1 ensures H3K27me3 deposition to precisely regulate the floral transition.

Size Restriction Is Required for Proper Functioning of a Bipartite Begomovirus AC4 Protein.

Many geminiviruses, including members of the genus Begomovirus, produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of more than 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus Tomato leaf curl Hsinchu virus contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to tomato leaf curl Hsinchu virus (ToLCHsV). The extended AC4 (EAC4) also showed a reduced ability to promote the infection of the heterologous virus Potato virus X than the wild-type AC4. When the wild-type AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from the abbreviated length, because it may lead to chloroplast localization. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rice Stripe Tenuivirus Has a Greater Tendency To Use the Prime-and-Realign Mechanism in Transcription of Genomic than in Transcription of Antigenomic Template RNAs.

Most segmented negative-sense RNA viruses employ a process termed cap snatching, during which they snatch capped RNA leaders from host cellular mRNAs and use the snatched leaders as primers for transcription, leading to the synthesis of viral mRNAs with 5' heterogeneous sequences (HSs). With traditional methods, only a few HSs can be determined, and identification of their donors is difficult. Here, the mRNA 5' ends of Rice stripe tenuivirus (RSV) and Rice grassy stunt tenuivirus (RGSV) and those of their host rice were determined by high-throughput sequencing. Millions of tenuiviral HSs were obtained, and a large number of them mapped to the 5' ends of corresponding host cellular mRNAs. Repeats of the dinucleotide AC, which are complementary to the U1G2 of the tenuiviral template 3'-U1G2U3G4UUUCG, were found to be prevalent at the 3' termini of tenuiviral HSs. Most of these ACs did not match host cellular mRNAs, supporting the idea that tenuiviruses use the prime-and-realign mechanism during cap snatching. We previously reported a greater tendency of RSV than RGSV to use the prime-and-realign mechanism in transcription with leaders cap snatched from a coinfecting reovirus. Besides confirming this observation in natural tenuiviral infections, the data here additionally reveal that RSV has a greater tendency to use this mechanism in transcribing genomic than in transcribing antigenomic templates. The data also suggest that tenuiviruses cap snatch host cellular mRNAs from translation- and photosynthesis-related genes, and capped RNA leaders snatched by tenuiviruses base pair with U1/U3 or G2/G4 of viral templates. These results provide unprecedented insights into the cap-snatching process of tenuiviruses.IMPORTANCE Many segmented negative-sense RNA viruses (segmented NSVs) are medically or agriculturally important pathogens. The cap-snatching process is a promising target for the development of antiviral strategies against this group of viruses. However, many details of this process remain poorly characterized. Tenuiviruses constitute a genus of agriculturally important segmented NSVs, several members of which are major viral pathogens of rice. Here, we for the first time adopted a high-throughput sequencing strategy to determine the 5' heterogeneous sequences (HSs) of tenuiviruses and mapped them to host cellular mRNAs. Besides providing deep insights into the cap snatching of tenuiviruses, the data obtained provide clear evidence to support several previously proposed models regarding cap snatching. Curiously and importantly, the data here reveal that not only different tenuiviruses but also the same tenuivirus synthesizing different mRNAs use the prime-and-realign mechanism with different tendencies during their cap snatching.

Structure and mechanism of the plant RNA polymerase V.

In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation pathway in plants. Here, we report on the structures of cauliflower Pol V in the free and elongation conformations. A conserved tyrosine residue of NRPE2 stacks with a double-stranded DNA branch of the transcription bubble to potentially attenuate elongation by inducing transcription stalling. The nontemplate DNA strand is captured by NRPE2 to enhance backtracking, thereby increasing 3'-5' cleavage, which likely underpins Pol V's high fidelity. The structures also illuminate the mechanism of Pol V transcription stalling and enhanced backtracking, which may be important for Pol V's retention on chromatin to serve its function in tethering downstream factors for RNA-directed DNA methylation.

Molecular basis of the plant ROS1-mediated active DNA demethylation.

Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.

Association between polymorphisms of collagen genes and susceptibility to intervertebral disc degeneration: a meta-analysis.

BACKGROUND: Collagens are important structural components of intervertebral disc. A number of studies have been performed for association between polymorphisms of collagen genes and risk of intervertebral disc degeneration (IVDD) but yielded inconsistent results. Here, we performed a meta-analysis to investigate the association of collagen IX alpha 2 (COL9A2) Trp2, collagen IX alpha 3 (COL9A3) Trp3, collagen I alpha 1 (COL1A1) Sp1 and collagen XI alpha 1 (COL11A1) C4603T polymorphisms with susceptibility to IVDD. METHOD: Eligible studies were retrieved by searching MEDLINE, EMBASE, Web of Science prior to 31 March, 2021. Odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated for association strength. RESULTS: A total of 28 eligible studies (31 datasets comprising 5497 cases and 5335 controls) were included. COL9A2 Trp2 carriers had an increased risk of IVDD than non-carriers in overall population (OR = 1.43, 95% CI 0.99-2.06, P = 0.058), which did not reach statistical significance. However, Trp2 carriers had 2.62-fold (95% CI 1.15-6.01, P = 0.022) risk than non-carriers in Caucasians. COL9A3 Trp3 was not associated with IVDD risk (OR = 1.28, 95% CI 0.81-2.02, P = 0.299). T allele and TT genotype of COL1A1 Sp1 (+ 1245G > T) were correlated with increased risk of IVDD. Significant associations were found between COL11A1 C4603T and IVDD risk under allelic (OR = 1.33, 95% CI 1.20-1.48), dominant (OR = 1.45, 95% CI 1.26-1.67), recessive (OR = 1.55, 95% CI 1.21-1.98) and homozygote model (OR = 1.81, 95% CI 1.40-2.34). CONCLUSIONS: COL1A1 Sp1 and COL11A1 C4603T polymorphism are associated with IVDD risk while the predictive roles of collagen IX gene Trp2/3 need verification in more large-scale studies.