Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana

Abstract

The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by apparent stabilization of pri-miRNAs. Whole-genome tiling array analyses reveal that se, abh1/cbp80, and cbp20 mutants also share similar splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, because introns retained in se, abh1/cbp80, and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for nonsense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1.

Fig. 1.

Requirement of the CBC for pri-miRNA processing. (A) Comparative gross morphology of 21-day-old wild-type (WT), abh1-285, abh1-753, and se-1 plants. (B) Detection of selected miRNAs by RNA blot in 10-day-old wild-type, abh1-285, abh1-753, and se-1 plants. Ethidium-stained gels on which tRNA and rRNA bands are visible are shown as loading control. (C) Comparative gross morphology of 21-day-old wild-type, se-1, abh1-285, and cbp20 plants. (D) Detection of selected miRNAs in 10-day-old wild-type, se-1, abh1-285, and cbp20 plants. (E) Real-time RT-PCR analysis of selected pri-miRNAs in wild-type, se-1, se-3, abh1-285, abh1-753, cbp20, and hyl1 plants. (F) Overlap of pri-miRNAs that accumulate to higher levels in abh1/cbp80-285, cbp20 and se-1 mutants. (G) As two examples for pri-miRNAs that accumulate in mutants, hybridization intensities on tiling arrays are shown for pri-miR160c and pri-miR172a. Values are averaged from three biological replicates each. Accumulation in se-1 is apparent for both pri-miRNA transcripts, but only pri-miR160c appears to be affected also by abh1 and cbp20 mutations. Tick marks indicate 100 bases each.

Fig. 2.

Splicing defects in abh1/cbp80-285, cbp20 and se-1 mutants. (A) Number of retained introns in mutants. (B) Overlap of retained introns among the three mutants examined. Parentheses give overlap expected by chance. (C) As one example for an improperly spliced mRNA detected by tiling arrays, hybridization signals of probes representing the AT3G01500 gene are shown for wild-type, abh1/cbp80, cbp20 and se plants. Values are averaged from three biological replicates each. Gray box highlights retained intron. Annotated splice forms (TAIR 7) are shown below, with blue indicating coding sequence. Tick marks indicate 500 bases each. (D) As a second example, tiling array signals for AT3G04670 are shown. Note second intron (purple underline) that is unchanged in the mutants. (E) Excerpt from table of genes with introns retained in abh1/cbp80, cbp20, and se-1 mutants (see Table S2). The second column (“Intron”) indicates intron position from 5′ end. As discussed in the text, first introns are overrepresented. FC, fold change. Details for highlighted genes are shown in F. (F) Validation of intron retention in selected genes by conventional RT-PCR analysis. gDNA, genomic DNA control. “+” and “−” indicates reactions with and without reverse transcriptase. The upper band corresponds to unspliced form (as in gDNA), the lower band to spliced form.

Fig. 3.

Analysis of splicing in other miRNA-processing mutants and in NMD mutants. (A) Comparative RT-PCR analysis of mRNAs with introns retained in abh1/cbp80, cbp20 and se mutants in other miRNA processing mutants and related genotypes (dcl1, dcl2 dcl3 dcl4, hyl1, hst and ago1). (B) Comparative RT-PCR analysis of NMD mutants (upf1 and upf3).

Fig. 4.

Accumulation of intron-containing pri-miRNAs. (A) Diagram of the pri-miRNA164a-1 transcript (53), with miRNA foldback indicated in gray, with intron-flanking and exon–exon-junction spanning oligonucleotide primers shown as arrows. RT-PCR results are shown on the right. (B) Diagram of the pri-miRNA156a transcript (52) and RT-PCR analysis. (C) Illustration comparing potential roles of CBC and SE in pri-miRNA processing and pre-mRNA splicing. SE might be part of a larger cap-binding complex (indicated by an additional component with a dashed outline) or might interact only transiently with the CPC.