A Comprehensive Map of Intron Branchpoints and Lariat RNAs in Plants.

Lariats are formed by excised introns, when the 5' splice site joins with the branchpoint (BP) during splicing. Although lariat RNAs are usually degraded by RNA debranching enzyme 1, recent findings in animals detected many lariat RNAs under physiological conditions. By contrast, the features of BPs and to what extent lariat RNAs accumulate naturally are largely unexplored in plants. Here, we analyzed 948 RNA sequencing data sets to document plant BPs and lariat RNAs on a genome-wide scale. In total, we identified 13,872, 5199, 29,582, and 13,478 BPs in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), and maize (Zea mays), respectively. Features of plant BPs are highly similar to those in yeast and human, in that BPs are adenine-preferred and flanked by uracil-enriched sequences. Intriguingly, ∼20% of introns harbor multiple BPs, and BP usage is tissue-specific. Furthermore, 10,580 lariat RNAs accumulate in wild-type Arabidopsis plants, and most of these lariat RNAs originate from longer or retroelement-depleted introns. Moreover, the expression of these lariat RNAs is accompanied by the incidence of back-splicing of parent exons. Collectively, our results provide a comprehensive map of intron BPs and lariat RNAs in four plant species and uncover a link between lariat turnover and splicing.

Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum.

Translational repression is a conserved mechanism in microRNA (miRNA)-guided gene silencing. In Arabidopsis, ARGONAUTE1 (AGO1), the major miRNA effector, localizes in the cytoplasm for mRNA cleavage and at the endoplasmic reticulum (ER) for translational repression of target genes. However, the mechanism underlying miRNA-mediated translational repression is poorly understood. In particular, how the subcellular partitioning of AGO1 is regulated is largely unexplored. Here, we show that the plant hormone brassinosteroids (BRs) inhibit miRNA-mediated translational repression by negatively regulating the distribution of AGO1 at the ER in Arabidopsis thaliana. We show that the protein levels rather than the transcript levels of miRNA target genes were reduced in BR-deficient mutants but increased under BR treatments. The localization of AGO1 at the ER was significantly decreased under BR treatments while it was increased in the BR-deficient mutants. Moreover, ROTUNDIFOLIA3 (ROT3), an enzyme involved in BR biosynthesis, co-localizes with AGO1 at the ER and interacts with AGO1 in a GW motif-dependent manner. Complementation analysis showed that the AGO1-ROT3 interaction is necessary for the function of ROT3. Our findings provide new clues to understand how miRNA-mediated gene silencing is regulated by plant endogenous hormones.

Identification of Intronic Lariat-Derived Circular RNAs in Arabidopsis by RNA Deep Sequencing.

Lariat RNAs are well-known by-products of pre-mRNA splicing in eukaryotes, which are produced by the excised introns when the 5' splice site (5' ss) joins with the branchpoint (BP) during splicing. In general, most of lariat RNAs are usually linearized by RNA debranching enzyme 1 (DBR1), followed by degradation for intron turnover. However, with the high-throughput RNA sequencing technology and bioinformatics methods, increasing evidences have shown that many lariat RNAs can stably accumulate under physiological conditions in both animals and plants. Here, we describe a large-scale analysis to systematically identify the lariat RNAs (i.e., intronic circular RNAs) in Arabidopsis by utilizing the RNA-sequencing data.

A lariat-derived circular RNA is required for plant development in Arabidopsis.

Lariat RNA is produced during pre-mRNA splicing, and it is traditionally thought as by-products, due to the quick turnover by debranching followed by degradation. However, recent findings identified many lariat RNAs accumulate with a circular form in higher eukaryotes. Although the remarkable accumulation, biological consequence of lariat-derived circular RNAs (here we name laciRNAs) remains largely unknown. Here, we report that a specific laciRNA from At5g37720 plays an essential role in plant development by regulating gene expression globally. We focus on 17 laciRNAs with accumulation in wild type plants by circular RNA sequencing in Arabidopsis. To determine biological functions of these laciRNAs, we constructed one pair of transgenic plants for each laciRNA, in which the local gene with or without introns was over-expressed in wild type plants, respectively. By comparing morphological phenotypes and transcriptomic profiles between two classes of transgenic plants, we show that over-expression of the laciRNA derived from the 1st intron of At5g37720 causes pleiotropic phenotypes, including curly and clustered leaf, late flowering, reduced fertility, and accompanied with altered expression of approximately 800 genes. Our results provide another example that a specific plant circular RNA regulates gene expression in a similar manner to that of other non-coding RNAs under physiological conditions.