Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses

Abstract

Long non-protein coding RNAs (npcRNA) represent an emerging class of riboregulators, which either act directly in this long form or are processed to shorter miRNA and siRNA. Genome-wide bioinformatic analysis of full-length cDNA databases identified 76 Arabidopsis npcRNAs. Fourteen npcRNAs were antisense to protein-coding mRNAs, suggesting cis-regulatory roles. Numerous 24-nt siRNA matched to five different npcRNAs, suggesting that these npcRNAs are precursors of this type of siRNA. Expression analyses of the 76 npcRNAs identified a novel npcRNA that accumulates in a dcl1 mutant but does not appear to produce trans-acting siRNA or miRNA. Additionally, another npcRNA was the precursor of miR869 and shown to be up-regulated in dcl4 but not in dcl1 mutants, indicative of a young miRNA gene. Abiotic stress altered the accumulation of 22 npcRNAs among the 76, a fraction significantly higher than that observed for the RNA binding protein-coding fraction of the transcriptome. Overexpression analyses in Arabidopsis identified two npcRNAs as regulators of root growth during salt stress and leaf morphology, respectively. Hence, together with small RNAs, long npcRNAs encompass a sensitive component of the transcriptome that have diverse roles during growth and differentiation.

Figure 1.

Expression patterns of four npcRNAs corresponding to new 24-nt siRNA precursors. (Left panels) The gray boxed regions (above or below are Watson and Crick strands, respectively) indicate the npcRNA gene and transcript (black line) on the Arabidopsis genomic DNA coordinates. Small RNAs identified in public databases are indicated by small bands on both strands. The size of each siRNA is indicated in Supplemental Figure 2; npcRNAs spanning small RNA loci (following a color code). (Middle panels) Expression of these npcRNAs in different tissues (roots, stems, flowers, cauline leaves, and rosette leaves) determined by qRT-PCR. Relative expression levels were normalized with ACT2 (AT3G18780), and values for roots were arbitrarily fixed to 1; SDs are shown. (Right panels) Expression in flowers or seedlings of Col-0 and a rdr2/6 or ago7 mutant. Values for Col-0 were fixed to 1.

Figure 2.

Regulation of genes in the RIBOCHIP. (A) Total number of genes that were either induced (red bars) or repressed (green bars) in each analyzed condition. Conditions include expression in roots versus leaves (roots), roots of phosphate starved plants (−P), plants under water stress (−H₂O), roots of plants treated with 150 mM NaCl (NaCl), and inflorescences of dcl1-9 mutants (dcl1). (B) Heat map showing expression level of each individual gene summarized in A. (*) npcRNAs identified by Hirsch et al. (2006); (**) npcRNAs identified in this study. Genes without asterisks correspond to additional regulated genes from the RIBOCHIP (annotation is indicated: coding sense counterparts of npcRNAs, RNA-binding proteins, miRNA precursors, and control genes).

Figure 3.

The npc83 is the miR869A precursor and is up-regulated in dcl4 mutants. The npc83 transcript encodes several small RNAs, notably miR869 (A), that are produced from a highly stable RNA-secondary stem structure (Z-score = 16). The position of the miRNA and detected siRNAs on the stem-loop of this npcRNA is indicated in B. (C) Expression of the MIR869A precursor was analyzed in dcl1-9, dcl2, dcl3, and dcl4 mutants and Col-0 seedlings by real-time RT-PCR. Data was normalized with ACT2 (AT3G18780), and values for Col-0 were arbitrarily fixed to 1. Two biological replicates gave similar results, and a representative example (SDs of technical replicates) is shown.

Figure 4.

Expression analysis of npcRNAs. (A) Accumulation of npc531 transcripts in dcl1 mutants. Real-time RT-PCR expression analysis of npc531 in wild-type and dcl1-9 inflorescences (A) was normalized with the ACT2 (AT3G18780) gene, and values for wild type were arbitrarily fixed to 1. On the right, predicted pairing between npc531 and miR319a. (B) Regulation of npc43, npc60, npc72, and npc536 expression. Northern analysis was performed on the indicated npcRNAs under different stress conditions (plants grown in phosphate starvation or under salt stress).

Figure 5.

Regulation of specific npcRNAs in different stress conditions. Real-time RT-PCR expression analysis of indicated npcRNAs in roots of plants grown in phosphate starvation (A) and after 3h and 24h in 150 mM NaCl (B). In every case data was normalized with ACT2 (AT3G18780) and values for non-treated or wild-type controls were arbitrary fixed to 1. For each cDNA synthesis, quantifications were made in triplicate and two biological replicates were analyzed. Values are means ± SD.

Figure 6.

Phenotypic and molecular analysis of transgenic plants overexpressing npc48. (A) Phenotype of Arabidopsis plants transformed with 35S∷npc48 construct. A wild-type (WT) plant (left) and two representative T1 35S∷npc48 plants (OEnpc48) from independent lines displaying a characteristic serrated leaf phenotype at different growth stages (central photos) are shown. Rosette leaves from WT and OEnpc 48 plants (lower left panel). Image of whole plants (right panel) illustrates the delayed flowering of OEnpc48 plants. (B) Expression analysis of npc48 and AGO1 mRNA from leaves of two npc48 overexpressing lines and control WT using real-time RT-PCR (left panel). Northern blot analyses of miR164, miR166, miR168, and U6 RNAs (right panel). Quantification is indicated setting the Col-0 value arbitrarily to 1 and normalizing to U6 values.

Figure 7.

Phenotypic and molecular analysis of transgenic plants overexpressing npcRNA536. Phenotype of Arabidopsis plants transformed with 35S∷npc536 constructs (Oenpc536). (A) Control and a representative OEnpc536 transgenic plants grown under the indicated salt stress conditions (0 mM, 100 mM, and 125 mM). (B) Quantification of primary root length and lateral root/primary root lengths in two independent transgenic OEnpc536 and control lines grown in 100 mM salt (n < 45 per experiment). SDs are indicated. These differences are statistically significant (t-test, 7 × 10⁻⁷), whereas no differences could be detected under normal growth conditions. (C) Schematic of the genomic positions and transcripts deriving from the npc536 (AT1G67920, white box) and AT1G67930 (dark gray box) loci. The nucleotide sequence of the overlapping region (light gray box) between the two transcripts is indicated.