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. P osttranscriptional gene regulation by microRNAs (miRNAs) is essential for the development and function of multicellular eukaryotes (1). In plants, miRNAs are processed from primary transcripts that contain partially complementary foldbacks of variable lengths (pri-miRNAs). Pri-miRNAs are similar to messenger RNAs (mRNAs) in being transcribed by DNA-dependent RNA polymerase II (pol II) and carrying a seven-methyl guanosine (m 7 G) cap at the 5Ј end and a polyadenosine (polyA) tail at the 3Ј end (1). Pri-miRNAs are processed to yield 20-to 22-nt-long mature miRNAs by an RNAseIII-like domain containing protein called DICER-LIKE1 (DCL1) (2-4). DCL1 interacts with the double-stranded (ds)RNA-binding protein HYPONASTIC LEAVES1 (HYL1) and the zinc finger protein SERRATE (SE) to ensure proper processing of pri-miRNAs (5-13). In common with dcl1 mutants, pri-miRNA levels are increased in plants that lack HYL1 or SE activity, whereas the amounts of mature miRNAs are reduced (5-9). All three proteins are found in nuclear processing centers, called D-bodies or SmD3/SmB nuclear bodies (12, 13).Mutants deficient in miRNA biogenesis suffer from a large range of morphological defects. Plants with null alleles of DCL1 or SE die as embryos, and even moderate reduction of DCL1 activity leads to a broad spectrum of developmental abnormalities (8,14,15). The weak se-1 allele causes only mild defects, including an alteration of phyllotaxis and the name-sake serrated leaves (16,17). It has been noted before that the se-1 phenotype is reminiscent of that of another mutant with impaired RNA metabolism, ABA hypersensitive 1 (abh1). Both mutants respond more strongly t...

Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis

“…In addition, SE may also act as a transcription mediator (Voisin et al, 2009). By analysis of public tiling array data sets, we found that se-1, se-3, cbp20-1, and cbp80-285 indeed accumulated higher levels of pri-miRNAs compared with the wild type (see Supplemental Figure 14 online), confirming previous results (Laubinger et al, 2008). Moreover, we found a global increase of lincRNA expression levels in these four mutants (see Supplemental Figure 14 online) with the following order of severity: cbp20, se-3, se-1, and cbp80.…”

“…To confirm this finding, we analyzed nine tiling array data sets obtained from different experiments performed by various groups (Laubinger et al, 2008(Laubinger et al, , 2010Zeller et al, 2009). Again, similar expression patterns appeared in all nine independent experiments that used various tissues and different stress treatments (see Supplemental Figure 9 online).…”

“…To investigate possible regulators involved in lincRNA biogenesis and processing, we applied this approach to analyze lincRNA expression levels as well as those of pri-miRNAs and mRNAs in tiling array data sets derived from three different organs and 11 mutant samples. These mutants were se-1, se-3, abh1-1/ cbp80-285, cbp20-1, upf1-1, upf3-1, dcl1-100, dcl2,3,4, ago1-25, hyl1-2, and ein5-6 Laubinger et al, 2008Laubinger et al, , 2010Kurihara et al, 2009aKurihara et al, , 2009b. Supplemental Figures 13 and 14 online compare accumulative frequency distributions of expression levels of lincRNAs, pri-miRNAs, and mRNAs between the wild type and each of the mutants.…”

“…This protein cooperates with HYL1 and DCL1 to process pri-miRNA (Yang et al, 2006), and along with ABH1/CBP80 and CBP20, it is required for mRNA and pri-miRNA splicing (Laubinger et al, 2008). The SEmediated splicing event is believed to prevent generation of related siRNAs to trigger posttranscriptional gene silencing .…”

“…In a genome-wide study, Laubinger et al (2008) detected 140 intron retention events (0.5%) out of 30,615 introns of pri-miRNAs and mRNAs in se, cbp20, and cbp80. Of the 36 lincRNAs annotated in TAIR9 as "other RNAs," four were co-upregulated by SE and CBPs and at least three lincRNAs contained introns (see Supplemental Data Set 14C online).…”