Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing

Abstract

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) -guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)-miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/ miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4-6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing.high-throughput sequencing | miRNA | trans-acting siRNA S mall RNAs, including microRNA (miRNA), several classes of endogenous small interfering RNA (siRNA), and Piwiassociated RNA (piRNA), direct silencing activities that shape transcriptomes and proteomes of eukaryotic organisms. miRNAs arise from transcripts containing self-complementary foldback structures that are initially processed to form 21-22nt miRNA/ miRNA* duplexes. In animals, primary transcripts with miRNA foldbacks (pri-miRNA) are processed first by the Microprocessor complex, which contains the RNase III-type protein Drosha and its cofactor Pasha (also known as DGCR8 in humans), then by Dicer, with partners that include the dsRNA-binding domain protein Loquacious (1). Plants orchestrate both pri-miRNA and pre-miRNA processing with the same (or very similar) complex, which includes the RNase-III like enzyme DICER-LIKE1 (DCL1) as the catalytic component (2-5). DCL1 interacts with the dsRNA binding protein HYPONASTIC LEAVES1 (HYL1) and the zinc-finger protein SERRATE (SE), both of which promote efficient and accurate miRNA biogenesis (2, 6-8).The transacting siRNA (tasiRNA) class represents a specialized type of amplification-dependent siRNA (9, 10). Primary tasiRNA-generating transcripts are first processed by miRNAguided cleavage (11,12). Either the 3′ (TAS1, TAS2 and TAS4 families) or 5′ (TAS3 family) cleavage product is stabilized and converted to dsRNA by RNA-DEPENDENT RNA POLY-MERASE6 (RDR6) (9, 10, 12, 13). Phased, 21-nt siRNAs are generated in register with the miRNA-guided cleavage site through sequential processing by DCL4. Routing of TAS3 precursor RNA requires two miRNA-guided events, both of which involve AGO7-miR390 complexes (14, 15). Interaction of AGO7-miR390 at a 3′ proximal target site results in primary transcript...

Evolution and Functional Diversification of MIRNA Genes

“…One feature was identified in mutants with defects in ath-MIR390a, ath-MIR172a, ath-MIR171a, ath-MIR167a, ath-MIR164c, and ath-MIR398a, with substitutions affecting the region 1 to 8 bp below the miRNA/miRNA* duplex and resulting in reduced or inaccurate miRNA accumulation (Cuperus et al, 2010b;Mateos et al, 2010;Song et al, 2010;Werner et al, 2010). For comparison, single nucleotide mutations in the loop-proximal region in many cases were considerably less deleterious, or had no effect, on miRNA accumulation (Mateos et al, 2010;Song et al, 2010;Werner et al, 2010).…”

“…For comparison, single nucleotide mutations in the loop-proximal region in many cases were considerably less deleterious, or had no effect, on miRNA accumulation (Mateos et al, 2010;Song et al, 2010;Werner et al, 2010). Mutations in the region below the miRNA/ miRNA* duplex that maintained wild-type structure were generally neutral, while mutations that opened or closed predicted bulges reduced the accumulation or altered the processing of the mature miRNAs (Cuperus et al, 2010b;Mateos et al, 2010;Song et al, 2010;Werner et al, 2010). In plant MIRNA foldbacks, this region is characterized by relatively weak base pairing, with a high number of one-to three-nucleotide bulges ( Figure 2B).…”

Mutagenesis for Crop Breeding and Functional Genomics

“…With smaller genome plants, it is possible to sequence whole genomes and clone genes by associating co-segregation of genotype to phenotype (Schneeberger et al 2009;Cuperus et al 2010). An approach known as MutMap has been described for cloning EMS-induced alleles in rice using a bulked segregant strategy, and the method further adapted so that alleles can be cloned without outcrossing (Abe et al 2012;Fekih et al 2013).…”

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers

“…Advances in DNA sequencing technologies have tremendously accelerated genetic mapping by combining bulk segregant analysis, that is, pooling recombinant genomes, with whole-genome sequencing, usually referred to as mapping by sequencing 2,3 . This approach is now becoming standard for mutation mapping and identification in many model species [3][4][5][6][7][8][9][10][11][12] and has even been applied to decipher quantitative traits with complex genetic architectures 13,14 . Recently, mutagen-induced changes have been used as novel markers, allowing mapping of mutations using isogenic mapping populations 10,15 .…”