The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum

Abstract

Background: Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms.

Results: Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions.

Conclusions: P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

Figure 1

Workflow of the small RNAs analysis in P. tricornutum. Top. Fragment lengths distribution of reads (histogram, center) is reported in a grey color scale distinguishing the five experimental conditions (LL, HL, NL, −Fe, D). The distribution of fragment location is also reported (pie chart, right) with a color scale indicating genes, intergenic regions, repeat regions, tRNA genes, ncRNAs and other loci. We distinguish two workflows described in boxes A and B, characterized by different local loci distributions of reads along the genome. (A) Sequence specific distribution of fragment lengths that is systematically observed for tRNA genes and intergenic regions. Reads were filtered in five steps, described in the 5 grey boxes. We obtained three main groups of results, indicated by squared boxes (number of predicted sRNAs is reported in parenthesis). The number of predicted sRNAs that were experimentally validated is also indicated, together with the experimental technique (NB, Northern Blot; PCR, Stem Loop PCR; H, sequencing data from [28]). (B) Distribution of fragment lengths that covers loci with overlapping reads and accumulated on both strands. This distribution pattern has been observed to either TEs or coding genes, associated to methylation. Examples of the periodic placement of sRNAs on three Codi LTR-retrotransposons on chromosome 31 and on a protein coding gene on chromosome 12 are reported. Color palette for TEs and genes is the same as above, and Highly Methylated regions are represented in purple.

Figure 2

A novel sRNA population of unknown biogenesis from an intergenic region. (A) Screenshot of the reads profile of the mature small RNA populations matching an intergenic region in the chromosome 2 (chr2: 29.039-29.123), with reads belonging to the five small RNA libraries. (B) Validation of the sRNAs expression by low molecular weight Northern blotting. Total RNA was extracted from cell cultures grown under different light conditions (High Light (HL), Normal Light (NL), Low Light (LL), Dark (D) and iron starvation (−Fe)) and Northern blots hybridized with radiolabeled probes recognizing the small 25 nt product and the U6 snRNA as reference loading control. M, microRNA Marker (New England Biolabs, USA).

Figure 3

A novel population of sRNAs derived from the U2 snRNA. (A). Screenshot of the reads profile associated to the U2 snRNA in chromosome 5 (chr5: 696.774–696.999). The U2-5′ (in orange) and U2-3′ (in green) sRNA populations associated to this region are indicated. (B) Validation of the U2-3′ expression by low molecular weight Northern blotting. Total RNA was extracted from cell cultures grown under different light conditions (High Light (HL), Normal Light (NL), Low Light (LL), Dark (D) and iron starvation (−Fe)) and Northern blot was probed using an oligonucleotide recognizing the U2-3′ sRNAs and the U6 snRNA as reference loading control. M, microRNA Marker (New England Biolabs, USA). (C) Relative transcript levels of U2-3′ and U2-5′ sRNAs determined by stem-loop qPCR, in cells grown under the same growth conditions described above. The expression values U2-3′ and U2-5′ were normalized to the snoRNA. Error bars are relative to three independent experiments. (D) Relative transcript levels of the U2 snRNA by qRT-PCR in cells grown under iron starvation and different light conditions. Normalization was done relative to histone H4 mRNA, used as a reference gene. Error bars are relative to three independent experiments. The U2 snRNA expression was also analyzed by Northern blot by using an oligonucleotide recognizing a region of the U2 snRNA upstream the U2-5′ small ncRNA, as a probe. (E) U2 snRNA secondary structure as annotated in Rfam [82] and the distribution of U2-5′ and U2-3′ highlighted on it. (F) Analysis of the U2-3′small RNAs in wild-type and overexpressing snRNA U2 lines (OE1 and OE2) by Northern blotting from cells grown under normal light condition. A total of 20 μg of RNA was loaded per lane and sRNA products were detected as described above.

Figure 4

sRNA associated to mature tRNAs. (A) tRNA structure and the four types of sRNA fragments associated to tRNAs (tRFs) as observed from the sequencing of the different libraries. Variations in fragment lengths are represented in light gray. (B) Expression validation of selected tRFs by Northern blotting. Total RNA was extracted from cell cultures grown under iron starvation (−Fe) and normal condition (+Fe) in Low Light. Northern blots were hybridized using oligonucleotides recognizing the small tRF products. M, microRNA Marker (New England Biolabs, USA).

Figure 5

Properties of sRNAs covering transposons. (A) Repartition of the sRNA sequences in the different diatom transposon families (only for TE annotation above 300 bp). We report the number of transposons with a sRNA coverage above 5 RPKM (grey) or with a low or no sRNA coverage (black). (B) 5′ end nucleotide composition of the reads (from position 1 to 5) aligning to transposons. (C) Relationship between sRNA coverage and autonomous TEs expression or autonomous TEs methylation. From left to right: transcript expression according to RNA-seq evidence, or cDNA libraries, and methylation status in transposons. (D) Experimental validation of the transcriptional activity level of autonomous transposons by qPCR. The transposons are grouped on the x-axis with an increasing coverage in sRNAs: low (sRNA RPKM < 5), middle (5 < RPKM < 10) and high (RPKM > 10) coverage. A grey shaded box is reported when the product could not be amplified. Autonomous transposons whose expression is supported by both EST and RNA-seq evidence are annotated with the symbols “++”. Labels above bins correspond to the Genbank identifier and localization of the transposons. (E) Comparison of methylation level and sRNA coverage on all Copia-type LTR transposons longer than 50 nt. Boxes are colored according to the length of the transposons region: 50 to 1000 nt (light grey), 1000 to 4000 nt (grey) and more than 4000 nt (dark grey). Methylation level is computed as the proportion of methylated probes that overlap the transposon region.