Hyponastic Leaves 1 protects pri-miRNAs from nuclear exosome attack

Abstract

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.

Keywords: HEN2; HYL1; SOP1; exosome; miRNA.

Fig. 1.

SOP1 mutations suppress the morphological defects of hyl1-2. (A) Vegetative phenotype of shy43, hyl1-2, and F1 plants from a cross between shy43 and hyl1-2. Aerial parts of 4-wk-old plants were photographed. (Scale bar, 1 cm.) (B) Manhattan plot showing the delta single nucleotide polymorphism (SNP) index between the shy43-like F2 pool and the hyl1-2 pool. SNP index was calculated by MutMAP (51). The causal gene was mapped to the left arm of Chr. 1, as highlighted by the black dotted circle. (C) Schematic structure of the SOP1 gene, the sop1-5 T-DNA insertion, and the sop1-6/sop1^shy43 mutation. The sop1-6 mutation and the resultant amino acid changes are highlighted in red. Open box, 5′ or 3′ untranslated regions; gray box, exons; solid line, introns; *, stop codon. (D) Sanger sequencing of the SOP1 cDNA from the Col-0 and sop1-6 plants. In sop1-6, the G-to-A mutation at acceptor site of the fourth intron of SOP1 resulted in a 1-bp shift of the splicing acceptor site, and consequently led to one guanine deletion. (E) Phenotype of different mutants and transgenic plants. Four-week-old plants and their rosette leaves of the indicated genotypes are shown. (Scale bar, 1 cm.) (F) sop1-5 rescues the morphological defects of hyl1-2. Four-week-old plants are photographed. (Scale bar, 1 cm.)

Fig. 2.

sop1-6 rescues a subset of miRNAs in hyl1-2. (A) Northern blot analysis of miRNA expression in Col-0, hyl1-2, and hyl1-2 sop1-6. U6 served as loading control. (B) qPCR analysis of the expression levels of miRNA target genes. ACTIN2 serves as an internal control. Another housekeeping gene UBQ5 was analyzed in parallel. Data are means of three biological replicates and error bars denote SD. Each dot point is the average value of three technical replicates. *P < 0.05; **P < 0.01 (paired t test). Inflorescence tissues of mixed stages were used in A and B. (C) Anther microsporangia phenotypes in Col-0 and different mutants. Numbers in the Lower Right corner of each panel indicate the number of anthers with shown phenotype vs. the total number of examined anthers.

Fig. 3.

Loop-to-base–processed miRNAs are preferentially restored in hyl1 sop1 and hyl1 hen2 mutants. (A) Twelve-day-old seedlings of Col-0, se-1, sop1-5, and se-1 sop1-5. White arrows indicate serrated leaf margins. (Scale bar, 1 cm.) (B) Twenty-five-day-old plants of Col-0, hyl1-2, hyl1-2 hen2-4, and hyl1-2 sop1-5. (Scale bar, 1 cm.) (C and D) Violin plots showing differential expression of miRNAs with different maturation ways in respective double mutants as compared with those in the hyl1-2 single mutant. Statistical differences between loop-to-base– and base-to-loop–processed miRNAs are calculated using two-tailed t test and shown as P values. Numbers on top of each plot indicate the numbers of miRNAs analyzed. See SI Appendix, Fig. S4 A and E for comparisons between mutants and wild type (i.e., Col-0). (E) Northern blot analysis of miRNA expression in different genotypes. Inflorescence tissues of mixed stages were used. Numerals indicate relative abundance of respective miRNAs relative to those in hyl1-2. U6 snRNA is a loading control. miRNAs generated from loop-to-base processing direction are bold and underlined.

Fig. 4.

Transcriptome analysis and accumulation of pri-miRNAs in different mutants. (A) Venn diagram showing the overlap of significantly up-regulated genes among different groups. Numbers in parentheses indicate the total number of up-regulated genes in the corresponding comparison group. (B) GO analysis (biological process [BP]) of 403 genes specifically up-regulated in hyl1-2 hen2-4 vs. hyl1-2 but not in hen2-4 vs. Col-0. (C) Violin plots showing differential expression of pri-miRNAs with different maturation ways in different mutants. Statistical differences between loop-to-base– and base-to-loop–processed pri-miRNAs are calculated using two-tailed t test and shown as P values. See also SI Appendix, Fig. S6G for comparisons between additional mutants and wild type. (D) Heatmap showing correlation between pri-miRNA overaccumulation and mature miRNA expression in hyl1-2 hen2-4 vs. hyl1-2. See SI Appendix, Fig. S6 H and I for comparisons of hyl1 sop1 vs. hyl1 and mutants vs. Col-0, respectively. (E) qPCR analysis of the expression levels of pri-miRNAs. The expression changes are normalized to those of ACTIN2, with expression levels in Col-0 set to 1. Data are means of three biological replicates and error bars denote SD. Each dot point is the average value of three technical replicates. *P < 0.05; **P < 0.01; ***P < 0.001 (paired t test). The Top shows schematic structures of pri-miRNAs and positions of primers relative to first cut sites. miRNA and miRNA* are highlighed in yellow and green, respectively. Red arrow heads depict first cut sites. Black half arrows indicate relative positions of primers used for qPCR analysis (not in scale).