Detection and Labeling of Small Non-Coding RNAs by Splinted Ligation.

Discovery and characterization of microRNAs (miRNAs) and other families of small RNAs lead researchers to study their structures/functions and their expression patterns. The splinted ligation method described here is based on nucleic acid hybridization. It is optimized for the direct labeling and quantitative detection of small RNAs. A specific bridge DNA oligonucleotide is used, which is perfectly complementary to both the target small RNA and a labeled ligation nucleic acid. The target RNA is subsequently labeled by ligation, detected by analysis in denaturing conditions, and quantified by phosphorimaging. The protocol does not require any specific material, and the procedure is fast and sensitive.

Simple sequence repeat analysis of new potato varieties developed in Alberta, Canada.

The worldwide demand for potato production requires the constant development of new potato varieties with improved yield, quality, disease resistance, and abiotic tolerance. However, cultivar registration is preceded by a long process to morphologically and physiologically characterize the plants. Notably, this process can be expedited by DNA marker analysis. Simple sequence repeats (SSRs), also known as microsatellites, are the most common reliable DNA markers used to discriminate between genotypes. In this study, 20 potato varieties, including five new genotypes developed in Alberta, Canada, were fingerprinted using 10 SSR markers selected for their high discriminatory power. Different SSRs were amplified from potato DNA using specific primers, and the DNA fragment sizes were analyzed by denaturing polyacrylamide gel electrophoresis. The number of alleles per locus ranged from two for the SSR marker STPoAc58 to six for STM0030 and STM0037 with an average of 4.4. In addition, a total of 77 unique patterns were observed for the 10 SSR markers. The polymorphic information content ranged from 0.477 to 0.802 with an average of 0.675 per locus. In this study, STM0037, STM1016, and STM1104 were found to be the best SSR markers to detect genetic differences between potato varieties. A minimum of two markers was required to distinguish between all 20 genotypes. Most importantly, this highly informative molecular tool confirmed that the developed potato varieties were genetically different from their respective maternal lines and potentially constituted new cultivars.

Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein.

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

Molecular Determinants for 23S rRNA Recognition and Modification by the E. coli Pseudouridine Synthase RluE.

The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis.

Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5.

In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)--the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U(55) in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.

RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature.

The box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Ψ) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyperthermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to Ψ conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity.

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs.

Detection and labeling of small non-coding RNAs by splinted ligation.

Discovery and characterization of microRNAs (miRNAs) and other families of small RNAs lead researchers to study their structures/functions and their expression patterns. The splinted ligation method described here is based on nucleic acid hybridization. It is optimized for the direct labeling and quantitative detection of small RNAs. A specific bridge DNA oligonucleotide is used, which is perfectly complementary to both the target small RNA and a labeled ligation nucleic acid. The target RNA is subsequently labeled by ligation, detected by analysis in denaturing conditions, and quantified by phosphorimaging. The protocol doesn't require any specific material, and the procedure is fast and sensitive.

Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.