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. 2012 Dec;24(12):4930-47.
doi: 10.1105/tpc.112.103697. Epub 2012 Dec 7.

LSM proteins provide accurate splicing and decay of selected transcripts to ensure normal Arabidopsis development

Carlos Perea-Resa  1 , Tamara Hernández-Verdeja, Rosa López-Cobollo, María del Mar Castellano, Julio Salinas
Affiliations

LSM proteins provide accurate splicing and decay of selected transcripts to ensure normal Arabidopsis development

Carlos Perea-Resa et al. Plant Cell. 2012 Dec.

Abstract

In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay.

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Figures

Figure 1.
Figure 1.
Expression Patterns of Arabidopsis LSM Genes. (A) Expression analysis of LSM genes in different organs of Arabidopsis by RNA hybridization using specific probes. Total RNA (20 µg) from 4-week-old rosette leaves (L), roots (R), flowers (F), and stems (S) was used. rRNA levels are shown as a loading control. (B) to (E) GUS activity in Arabidopsis plants containing the fusion LSM8pro-GUS. Whole plant (B), root (C), cross section of a stem (D), and flower (E).
Figure 2.
Figure 2.
Subcellular Localization of Arabidopsis LSM Proteins. (A) to (C) Subcellular localization of different LSM-GFP proteins in root tip cells from 6-d-old Arabidopsis seedlings. Seedlings grown under control conditions (A), seedlings grown under control conditions and subsequently exposed 2 h at 37°C (B), and seedlings grown under control conditions and subsequently exposed 2 h at 37°C with cycloheximide (CHX) (C). Bars = 10 µm. (D) Colocalization of LSM1A-GFP and LSM1B-GFP with RFP-DCP1 in root tip cells from 6-d-old Arabidopsis seedlings grown under control conditions (top panel) and subsequently exposed 2 h at 37°C (bottom panel). Bars = 10 µm. (E) Subcellular localization of GFP-DCP2 and GFP-VCS in root tip cells from 6-d-old wild-type (WT) and lsm1a lsm1b Arabidopsis seedlings grown under control conditions and subsequently exposed 2 h at 37°C. Bars = 10 µm.
Figure 3.
Figure 3.
Organization of Arabidopsis LSM Proteins. (A) Cellular model showing cytoplasmic and nuclear heptameric LSM complexes as described in yeast and humans. (B) and (C) Visualization of in vivo interactions between Arabidopsis LSM proteins by BiFC assays. The corresponding LSM-nGFP/LSM-cGFP proteins were pairwise tested by Agrobacterium tumefaciens–mediated transformation in N. benthamiana leaves. Interactions between LSM1A/LSM2, LSM1A/LSM4, LSM1A/LSM8, LSM8/LSM2, LSM8/LSM4, and LSM2/LSM4 (B), and LSM2/LSM3A, LSM6A/LSM3A, LSM6A/LSM5, LSM5/LSM7, LSM7/LSM4, LSM2/LSM7, LSM4/LSM6A, LSM6A/LSM7, and LSM3A/LSM5 (C) are presented. Bars = 20 µm. (D) Subcellular localization of LSM4-GFP in root tip cells from 6-d-old wild-type (WT), lsm8-1, and lsm1a lsm1b Arabidopsis seedlings grown under control conditions. Bars = 10 µm.
Figure 4.
Figure 4.
Phenotypic Analysis of lsm1a lsm1b Double Mutant. (A) Schematic representation of lsm1a and lsm1b T-DNA insertions in LSM1A and LSM1B genes, respectively. Boxes symbolize exons. (B) Expression analysis of LSM1A and LSM1B genes in 2-week-old wild-type (WT), lsm1a, lsm1b, and lsm1a lsm1b Arabidopsis plants by RNA hybridization using specific probes. rRNA levels are shown as a loading control. (C) to (K) Morphological phenotypes of wild-type, lsm1a lsm1b, and c-lsm1a plants. Three-day-old seedlings (C), 5-d-old seedlings (D), cotyledon vein patterns (E), rosette leaves (F), cauline leaves (G), 12-d-old seedlings (H), 6-week-old plants (I), siliques (J), and seeds (K).
Figure 5.
Figure 5.
Phenotypic Analysis of lsm8 Mutants. (A) Schematic representation of lsm8-1 and lsm8-2 T-DNA insertions in the LSM8 gene. Boxes symbolize exons. (B) Expression analysis of LSM8 in 2-week-old wild-type (WT), lsm8-1, and lsm8-2 Arabidopsis plants by RNA hybridization using a specific probe. rRNA levels are shown as a loading control. (C) to (I) Morphological phenotypes of wild-type, lsm8-1, lsm8-2, and c-lsm8 plants. Five-day-old seedlings (C), cotyledon vein patterns (D), rosette leaves (E), 12-d-old seedlings (F), 6-week-old plants (G), siliques (H), and seeds (I).
Figure 6.
Figure 6.
mRNA Stability and Accumulation of Capped Transcripts in the lsm1a lsm1b Double Mutant. (A) to (D) Transcript accumulation in lsm1a lsm1b and c-lsm1a plants. Levels of several transcripts in 6-d-old Arabidopsis seedlings of the wild type (WT) and lsm1a lsm1b ([A] and [B]) and of the wild type and c-lsm1a ([C] and [D]) at different minutes after cordycepin treatment. (A) and (C) RNA hybridizations using specific probes. rRNA levels were used as a loading control. The estimated half-life (min) of mRNAs is shown to the right of each panel (wild type/analyzed genotype). (B) and (D) Normalized quantification of the hybridization bands corresponding to genes of (A) (shown in [B]) and (C) (shown in [D]). (E) Accumulation of capped transcripts corresponding to different genes in 6-d-old wild-type, lsm1a lsm1b, c-lsm1a, and c-lsm1b Arabidopsis seedlings by RACE-PCR. RACE-PCR products obtained using a low (left panel) and high (right panel) number of cycles are shown. The products of EIF4A1, also derived from RACE-PCR, were used as a loading control.
Figure 7.
Figure 7.
Intron Retention and U6 snRNA Stability in lsm8 Mutants. (A) Validation of intron retention events in different genes identified by tiling arrays in the lsm8-1 mutant. RT-PCR was performed with total RNA from 2-week-old wild-type (WT), lsm8-1, lsm8-2, c-lsm8, and lsm1a lsm1b Arabidopsis plants and specific pairs of primers for each gene. In all cases, one primer was situated inside the retained intron and the other in an adjacent exon. Genomic DNA (Genomic) was used as a control. +RT indicates reactions with reverse transcriptase (RT). Control reactions without RT (−RT) were also performed. TUBULIN expression is shown as a loading control. (B) and (C) Stability of U6 snRNA in lsm8-1, lsm8-2, and c-lsm8 plants. Levels of U6 snRNA, U3 snoRNA, and U4 snRNA in 6-d-old Arabidopsis seedlings of the wild type, lsm8-1, and lsm8-2 (B) and of the wild type and c-lsm8 (C) at different hours after cordycepin treatment, as shown by RNA hybridization using specific probes. rRNA levels were used as a loading control.
Figure 8.
Figure 8.
Accumulation of Development-Related Transcripts in the lsm1a lsm1b Double Mutant. (A) and (B) Expression levels of different developmental genes detected in the microarray with altered expression in lsm1a lsm1b. The relative levels of 12 RNAs that in the microarray were increased (A) or decreased (B) are shown. Real-time RT-PCR analyses were performed with total RNA from 2-week-old wild-type (WT), lsm1a lsm1b, c-lsm1a, and c-lsm1b Arabidopsis plants and specific pairs of primers for each gene. (C) Accumulation of transcripts corresponding to several developmental genes detected in the microarray with increased expression in lsm1a lsm1b. In all cases, the relative transcript levels were determined by real-time RT-PCR analysis, as described above, in wild-type, lsm1a lsm1b, c-lsm1a, and c-lsm1b Arabidopsis plants at different minutes after cordycepin treatment. Values are relative to the control values obtained for each genotype. (D) Accumulation of capped transcripts corresponding to genes analyzed in (C) in 2-week-old wild-type, lsm1a lsm1b, c-lsm1a, and c-lsm1b Arabidopsis plants by RACE-PCR. RACE-PCR products obtained using a low (left panel) and high (right panel) number of cycles are shown. The products of EIF4A1, also derived from RACE-PCR, were used as a loading control.
Figure 9.
Figure 9.
Intron Retention in Developmental Genes in lsm8 Mutants. (A) and (B) Expression levels of different development-related genes detected in the tiling array with altered expression in lsm8 mutants. The relative levels of 11 RNAs that were increased (A) or decreased (B) in the array are shown. Real-time RT-PCR analyses were performed with total RNA from 2-week-old wild-type (WT), lsm8-1, lsm8-2, and c-lsm8 Arabidopsis plants and specific pairs of primers for each gene. (C) Validation of intron retention events in some developmental genes identified by tiling arrays in lsm8-1. RT-PCR was performed with total RNA from 2-week-old wild-type, lsm8-1, lsm8-2, and c-lsm8 Arabidopsis plants and specific pairs of primers for each gene. In all cases, one primer was situated inside the retained intron and the other in an adjacent exon. Genomic DNA (Genomic) was used as a control. +RT indicates reactions with reverse transcriptase (RT). Control reactions without RT (−RT) were also performed. TUBULIN expression is shown as a loading control.
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