SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

Abstract

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.

Figure 1. Description of the SINE-induced phenotype.

Different individuals from the Col0-SB1.7(18) transgenic line producing SB1 RNA are compared to wild type (Col0) plants. A. Impact on root growth. Col0-SB1.7(18) individuals have much shorter roots compared to the wild type. Six different Col0-SB1.7(18) individuals representing the variability in this line are presented B. Comparison of 27 days seedlings from Col0, Col0-SB1.7(18) and hyl1-2, drb4-1 RNAi mutant lines. Leaves from Col0-SB1.7(18) individuals are narrower, irregular in shape, and present a downward curvature. Three different Col0-SB1.7(18) individuals representing the variability in this line are presented. C. Col0-SB1.7(18) individuals have a general delayed growth, present shorter siliques and suffer from a partial loss of apical dominance. Two different Col0-SB1.7(18) individuals representing a mild and a severe case are presented. D. Typical pattern of SB1.7 SINE expression obtained by PAA gel hybridization after an 18h exposure. Three SB1.7-specific RNA species were detected, as expected from the post-transcriptional processing of the SINE primary transcript . The sizes (in nucleotides) of the hybridizing SINE RNA species are shown. Following stripping of the probe, the membrane was re-hybridized with a U6-specific probe and exposed for 10 min.

Figure 2. SINE RNA can bind to a subset of dsRBPs.

A. Gel retardation experiments using GST, a recombinant GST-HYL1 fusion protein and [α-³²P]-labeled single-stranded RNA, perfect double-stranded RNA, SB1.7 RNA, single-stranded DNA and double-stranded DNA B. Gel retardation experiments using GST, a recombinant GST-DRB4 fusion protein and [α-³²P]-labeled single-stranded RNA, perfect double-stranded RNA, SB1.7 RNA, single-stranded DNA and double-stranded DNA. In both cases the amount of recombinant proteins used is indicated (in µM).

Figure 3. Protection from RNase V1 digestions of SINE RNA by two different dsRBPs.

Prior to RNase V1 digestion, in vitro transcript of SB1 was subjected to protection by increasing concentrations of expressed dsRBPs: Dr2 (the second dsRBD of Xenopus laevis ADAR1), HYL1, combination of Dr2 and HYL1, or DRB4. Regions protected by Dr2 and HYL1 are marked alongside short run gel (A), long run gel (B) and predicted folding pattern (C) by brown and green bars, respectively. Three independent experiments gave similar results as the one presented. Nucleotides marked with asterisks seem to adopt more prominent helical structure upon protein binding and, therefore, become more prone to RNase V1 cleavage. DRB4 is showing no effect on RNase V1 cleavage, confirming its low in vitro binding affinity to SB1 RNA. Dr2 and HYL1, in this case, bind to and protect similar regions of RNA. HYL1 is, however, showing stronger binding affinity than Dr2. (HL) represents a partially hydrolyzed RNA ladder. Denaturating RNase A and T1 digests give the position of pyrimidine and G residues respectively. The control lane shows untreated RNA samples and the (0) lane represent RNase V1 digestion without recombinant proteins added. A labeled 23-mer oligoribonucleotide was also loaded on the gel to help in band size determination.

Figure 4. Protection from RNase V1 digestion of SELEX clone 11Dr2(7) by different dsRBPs.

Prior to RNase V1 digestion, in vitro transcript of SELEX clone 11Dr2(7) was subjected to protection by increasing concentrations of expressed dsRBPs: Dr2, HYL1, combination of Dr2 and HYL1, or DRB4. Regions protected by Dr2 and HYL1 are marked alongside short run gel (A), long run gel (B) and predicted folding pattern (C) by brown and green bars, respectively. Three independent experiments gave similar results as the one presented. Nucleotides marked with asterisks seem to adopt more prominent helical structure upon protein binding and, therefore, become more prone to RNase V1 cleavage. DRB4 is showing no effect on RNase V1 cleavage of given RNA, confirming its low binding affinity for imperfect RNA duplexes. (HL) represents a partially hydrolyzed RNA ladder. Denaturating RNase A and T1 digests give the position of pyrimidine and G residues respectively. The control lane shows untreated RNA samples and the (0) lane represent RNase V1 digestion without recombinant proteins added. A labeled 23-mer oligoribonucleotide was also loaded on the gel to help in band size determination.

Figure 5. Molecular impact of SINE transcription on the different mi/tasiRNA pathways.

A. Molecular impact on the miRNA pathway. Examples of five miRNAs that accumulate to lower levels in flowers from SINE expressing individuals (Col0-SB1.7(18)) compared to wild type (Col0). The correlative increase of the miR171-targeted SCL6-III mRNA is shown. B. Molecular impact on the tasiRNA pathway. The two miRNAs known to prime the synthesis of the tasiRNA precursors (miR173 for TAS1 and 2 and miR390 for TAS3) accumulate to a lower level in flowers from SINE expressing individuals (Col0-SB1.7(18)) compared to wild type (Col0). Consequently mature tasiRNA products (5′D7(+) TAS3, siR255 TAS1) are less abundant in SINE expressing individuals and messenger RNA targets of TAS3 (ARF3 and ARF4 mRNAs) are over-represented. C. The accumulation of the DCL4-DRB4-dependent miR822 is unchanged in the Col0-SB1.7(18) transgenic line but miR822 is undetectable in the drb4 mutant line. The relative proportion of miRNA, tasiRNA and mRNAs (the mean of at least three experiments) normalized using the U6 RNA or Actin mRNA signal is indicated. Similar results were obtained using the Col0-SB1.7(4) transgenic line.