Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control - PubMed (original) (raw)

Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control

Yuling Jiao et al. Mol Syst Biol. 2010.

Abstract

Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Detection and quantification of cell-specific genes. (A) Diagrams showing the spatiotemporal domains being profiled. Sepal (se) primordia are visible at stage 4, and sepal, petal (pe), stamen (st) and carpel (ca) primordia are all visible at stages 6–7. (B) Translated mRNAs for AP1, AP3, and AG domains at flower stages 6–7 for a 1.0-kb region of chromosome-1 containing RBE, which is specific for AP1 and AP3 domains, and (C) a 4.2-kb region of chromosome-1 containing APL, which is highly expressed in the AG domain. TAIR annotated transcripts are shown as gray boxes at the bottom of (B) and (C) with ORFs highlighted as thick boxes. TAIR has two annotations for APL with the second form only detected in the AG domain. Reads covering exon–exon junctions are highlighted by short lines in (C). (D) Expression of previously characterized flower-specific genes. Genes were identified manually by searching PubMed abstracts followed by manual summarization of in situ hybridization data from each publication. Rows and columns were ordered manually. Relative expression levels were calculated by comparing each domain-specific expression with average expression across all domains. (E) Venn diagram of the cell domain-enriched genes that exhibited significant (⩾two-fold with P<0.001) up-regulation as compared with other domain(s) at stage 4. The numbers in middle areas indicate genes without domain-specific expression. (F) Count of genes with expression (RPKM ⩾1) for each spatiotemporal cell domain. (G) Differentially expressed (⩾two-fold with P<0.001) genes between stages 6–7 and stage 4 in each cell domains.

Figure 2

Figure 2

The spatiotemporally regulated transcriptome during early flower development. (A) GO analysis identifies significantly overrepresented (P<0.001) gene categories under ‘Biological Process’ for the cell-specific transcripts in the AP3 domain at stages 6–7. Color bar: significance level for categories by hypergeometric test with FDR correction. (B) Domain-specific enrichment of hormone-responsive genes in each floral domain at stages 4 or 6–7. Red for upregulated genes and blue for downregulated genes. (C) Cell-specific enriched known _cis_-elements at flower stages 4 and 6–7. Candidate cis- and _trans_-transcriptional control cognate partners are shown in the blue box, in which GATA promoter motif and C2C2-GATA family transcription factors are enriched in floral domains. Only significantly overrepresented (P<0.001) classes by hypergeometric test with FDR correction are colored in (B) and (C). (D) Number of genes with splicing isoforms differentially expressed between any two floral domains at stages 4 and 6–7.

Figure 3

Figure 3

Intron retention events detected during flower development. (A) Expression profiles of detected TAIR-annotated introns. Red dots represent introns detected in only total poly(A)+ mRNAs, whereas blue and yellow dots represent introns detected in both total and translating mRNAs, or only translating mRNAs. (B) Transcript levels are negatively correlated with IR levels (_R_=−0.76, P<10−23). IR levels are quantified by normalizing detected intron levels with neighboring exon levels. (C) Intron subtypes significantly differ in IR levels. Percentages of IR events for intron subtypes are shown. Number of introns for each subtype is included below subtype name. (D) GO analysis identifies significantly overrepresented (P<0.001) gene categories under ‘Developmental Process’ for the genes with IR events in floral organs at flower stage 4. Color bar: significance level for categories by hypergeometric test with FDR correction.

Figure 4

Figure 4

Translational regulation during flower development. (A) M-A plot in which log2-transformed ratios of translating versus total mRNA signal for stage 4 flowers are plotted against log-intensity averages. Transcripts with statistically different levels in the translating and total RNA populations (P<0.001) and a ratio above two are considered as highly translated and are shown in red. Transcripts with statistically different levels and ratio below 0.5 were considered as weakly translated and are shown in green. Plants expressing HF–RPL18 under the RPL18 promoter were used for translatome profiling. (B) Biases around the start codon shown as the fractions of bases. At each base (rectangle), the left half represents the fraction of each base in highly translated genes and the right half represents the fraction of each base in weakly translated genes. Enriched bases are labeled. (C) Enhanced translation for short transcripts. A transcript length distribution of all expressed genes is shown by the histogram and the black line, while red and green lines show distributions of highly and weakly translated transcripts, respectively. (D) Relationship between gene function and translation levels. All genes detected as expressed for ‘Biological Process’ categories (Total) are compared with highly translated (red) and weakly translated (green) transcripts. ‘Distribution’ refers to the percentages of genes annotated to descriptive terms in a particular GO category divided by all genes. Categories with FDR corrected hypergeometric test P<0.001 are marked with stars. (E) Estimated regulation modes of miRNA target genes. Scatterplot of uncapping levels (endonucleolytic cleavage) of miRNA and ta-siRNA (TSA) target mRNAs versus translation efficiency (translation inhibition) of the same mRNAs in stage 4 flower tissues. Targets of miR172, including AP2 and _AP2_-like mRNAs are highlighted in red.

Figure 5

Figure 5

Polysome-associated ncRNAs. (A) Summary of numbers and percentages of detection for ncRNAs, TE-related RNAs, pseudogene RNAs, and RNAs from TARs in stage 4 flower tissues. The vertical line separates genes observed associated with ribosomes, or not, in our analysis. Additional genes with detection in total mRNA population are labeled as ‘w/transcription’. Polysome-associated transcripts supported by proteomics data (Baerenfaller et al, 2008; Castellana et al, 2008) are labeled as ‘w/peptide’. Percentages of polysome-associated transcripts for each category are listed in parentheses. Number of nonpolysome-associated TE-related genes or pseudogenes is listed at the right end of each bar. (B) Most polysome-associated ncRNAs lack coding capacity. A RFC score distribution of all these ncRNAs is shown by the histogram, while red and blue lines show distributions of conserved intergenic regions and protein-coding genes (with UTRs) within the similar length range of the ncRNAs, respectively. RFC scores were calculated as described previously (Clamp et al, 2007) based on alignments between the Arabidopsis thaliana TAIR9 build and Arabidopsis lyrata v1.0 release build using the VISTA pipeline (Frazer et al, 2004).

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