STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis

Abstract

MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1 (STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesis-defective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1 is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level.

Figure 1.

Phenotype comparisons between sta1-1 and miRNA biogenesis defective mutants. (A) Leaf and silique morphology. sta1-1 showed serrated leaves and small siliques similar to miRNA biogenesis mutants. (B) Silique phyllotaxy defects in sta1-1 and miRNA biogenesis mutants. Arrow heads indicate altered silique phyllotaxy.

Figure 2.

Reduction of miRNA levels in sta1-1. (A) Small RNA sequencing analysis of miRNAs in WT and sta1-1. From the small RNA sequencing results, abundance of miRNAs in sta1-1 was compared with WT. (B) RNA blot hybridization of miRNAs from 4-week-old Col-0, se-1, hyl1-2 and sta1-1. The levels of U6 small nuclear RNA were shown as loading controls. Numbers below the blot images are relative intensities of the miRNA bands.

Figure 3.

qRT-PCR expression analysis of selected miRNA target transcripts in Col-0, se-1, hyl1-2 and sta1-1. Relative amounts of mRNA levels were obtained by dividing the expression level of the mutant with WT value. Three biological samples were used.

Figure 4.

qRT-PCR expression analysis of selected pri-miRNA transcripts in Col-0, se-1, hyl1-2 and sta1-1. Relative amounts of mRNA levels were obtained by dividing the expression level of the mutant with the WT value. Three biological samples were used.

Figure 5.

Intron-retention analysis of selected pri-miRNA. (A–G) Analysis of retained intron in Col-0, se-1, hyl1-2 and sta1-1. Five intron-containing pri-miRNAs were investigated by RT-PCR. Splicing pattern of pri-miR160b (A), pri-miR166a (B), pri-miR166b (C), pri-miR172a (D), pri-miR172b (E) and pri-miR166a (F). Tubulin gene was used as a loading control (G). Asterisk, unspliced pri-miRNA; arrow-head, splicing intermediate; arrow, fully spliced or mature pri-miRNA. (H) Patterns of processing intermediates of pri-miR172b in Col-0, se-1, hyl1-2 and sta1-1. RT-PCR fragments of pri-miR172b in each genotype were cloned and sequenced to compare the retention patterns.

Figure 6.

Comparison of transcript levels of miRNA biogenesis genes in Col-RD29A-LUC and sta1-1. (A) RT-PCR results of miRNA biogenesis genes. DCL1 transcript levels were lower in sta1-1 than Col-RD29A-LUC. (B) qRT-PCR results of miRNA biogenesis genes. qRT-PCR confirmed that reduced levels of DCL1 transcript in sta1-1. Three replicates were used.

Figure 7.

A model for STA1 function in miRNA biogenesis. SE involvement in pre-mRNA processing was previously reported (57). In addition to mRNA splicing, STA1 is also involved in splicing of intron-containing pri-miRNAs. STA1 has a direct role in pri-miRNA splicing that affects miRNA processing and also indirectly affects intronless pri-miRNA processing by modulating the DCL1 transcript levels. It is not clear that STA1 regulation on the DCL1 transcript levels occurs through the STA1-including splicing complex or other STA1 unique functions.