Biology subjects

Lunardon, A.

Publications and source records attributed to Lunardon, A..

4 recordsLinked to original sources

bioRxiv2024

The Chlamydomonas reinhardtii CLiP2 mutant collection expands genome coverage with high-confidence disrupting alleles

Chlamydomonas reinhardtii (Chlamydomonas hereafter) is a powerful model organism for studies of photosynthesis, ciliary motility, and other cellular processes [1-4]. The CLiP library of mapped nuclear random insertion mutants [5,6] has accelerated progress for hundreds of laboratories in these fields by providing mutants in genes of interest. However, its value was limited by its modest coverage of the genome with high-confidence disruption alleles (46% of nuclear protein-coding genes with 1+ high-confidence allele in exons/introns; 12% of genes with 3+ alleles in exons/introns). Here we introduce the CLiP2 (Chlamydomonas Library Project 2) library, which greatly expands the number of available mapped high-confidence insertional mutants. The CLiP2 library includes 71,700 strains, covering 79% of nuclear protein-coding genes with 1+ high-confidence allele in exons/introns and 49% of genes with 3+ alleles in exons/introns. The mutants are available to the community via the Chlamydomonas Resource Center.

plant biology↗

bioRxiv2021

Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs in Solanum lycopersicum and Nicotiana benthamiana

Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach; they have the advantage of producing just a single functional small RNA which can be designed for high target specificity and low off-target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans-acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA- and tasiRNA-based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5-most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3 end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single-target assays such as RNA blots or real-time PCR, is also discussed. Significance statementSeveral strategies for multiplexed gene silencing using artificial microRNAs or tasiRNAs have been described. We find that many result in imprecise processing, and thus low accumulation of the intended small RNAs. Our findings highlight the importance of small RNA sequencing to fully analyze gene silencing experiments, and also the need for continued methodological development of these methods.

plant biology↗

bioRxiv2019

Integrated annotations and analyses of small RNA-producing loci from 47 diverse plants

Plant endogenous small RNAs (sRNAs) are important regulators of gene expression. There are two broad categories of plant sRNAs: microRNAs (miRNAs) and endogenous short interfering RNAs (siRNAs). MicroRNA loci are relatively well-annotated but comprise only a small minority of the total sRNA pool; siRNA locus annotations have lagged far behind. Here, we used a large dataset of published and newly generated sRNA sequencing data (1,333 sRNA-seq libraries containing over 20 billion reads) and a uniform bioinformatic pipeline to produce comprehensive sRNA locus annotations of 47 diverse plants, yielding over 2.7 million sRNA loci. The two most numerous classes of siRNA loci produced mainly 24 nucleotide and 21 nucleotide siRNAs, respectively. 24 nucleotide-dominated siRNA loci usually occurred in intergenic regions, especially at the 5-flanking regions of protein-coding genes. In contrast, 21 nucleotide-dominated siRNA loci were most often derived from double-stranded RNA precursors copied from spliced mRNAs. Genic 21 nucleotide-dominated loci were especially common from disease resistance genes, including from a large number of monocots. Individual siRNA sequences of all types showed very little conservation across species, while mature miRNAs were more likely to be conserved. We developed a web server where our data and several search and analysis tools are freely accessible at http://plantsmallrnagenes.science.psu.edu.

plant biology↗

bioRxiv2018

Several phased siRNA annotation methods can produce frequent false-positives for 24 nucleotide RNA-dominated loci in plants

Small RNAs regulate key physiological functions in land plants. Small RNAs can be divided into two categories: microRNAs (miRNAs) and short interfering RNAs (siRNAs); siRNAs are further sub-divided into transposon/repetitive region-localized heterochromatic siRNAs and phased siRNAs (phasiRNAs). PhasiRNAs are produced from the miRNA-mediated cleavage of a Pol II RNA transcript; the miRNA cleavage site provides a defined starting point from which phasiRNAs are produced in a distinctly phased pattern. 21-22 nucleotide (nt)-dominated phasiRNA-producing loci (PHAS) are well represented in all land plants to date. In contrast, 24 nt-dominated PHAS loci are known to be encoded only in monocots and are generally restricted to male reproductive tissues. Currently, only one miRNA (miR2275) is known to trigger the production of these 24 nt-dominated PHAS loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt-dominated PHAS loci also exist in Arabidopsis thaliana. We find that highly expressed heterochromatic siRNAs were consistently mis-identified as 24 nt-dominated PHAS loci using multiple PHAS-detecting algorithms. We also find that MIR2275 is not found in A. thaliana, and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used PHAS-detecting algorithms which may lead to false positives when trying to annotate new PHAS, especially 24 nt-dominated loci.

plant biology↗