Figure 1.
Illumina/Solexa paired-end sequencing and data analysis.
(A) The strategy for the Illumina/Solexa paired-end sequencing library preparation. (B) 6% denaturing urea PAGE of RNA samples and 6% native PAGE of DNA samples for the Illumina/Solexa paired-end sequencing library preparation according to the flowchart shown in (A). (C) The genome location distribution of contigs assembled from the Illumina/Solexa paired-end sequencing reads. “Annotated ncRNA” represents ncRNAs in PlasmoDB 10.0, including rRNAs, tRNAs, snoRNAs, U1–U6 snRNAs, SRP RNA, RNAse P, and RNAse MRP RNAs. “Other reported studies” denotes the ncRNAs that were described in the five studies by Mourier T et al. (2008), Mishra PC et al. (2009), Raabe CA et al. (2009), Otto TD et al. (2010) and Broadbent KM et al. (2011) but not in PlasmoDB 10.0. (D) The size distribution of all the 1,198 novel is-ncRNAs. (E) All P. falciparum is-ncRNAs identified to date. “Previously known is-ncRNAs” denotes all the is-ncRNAs annotated in PlasmoDB 10.0 and reported is-ncRNAs in the five studies.
Table 1.
Illumina/Solexa paired-end sequencing mapping to the P. falciparum genome and the distribution of assembled contigs.
Figure 2.
Conservation analysis of the novel is-ncRNAs.
Conservation analysis of is-ncRNAs based on the phastCons score distributions across eight Plasmodium species: P. falciparum, P. reichenowi, P. gallinaceum, P. vivax, P. knowlesi, P. yoelii, P. berghei, and P. chabaudi. The white dots indicate the median phastCons scores of the RNAs in each class, and the bold black lines indicate the interquartile range.
Figure 3.
Structure-based analysis of the novel intergenic is-ncRNAs.
(A) Clustering of the 313 novel intergenic is-ncRNAs and 141 known functional ncRNAs with intermediate sizes based on their predicted secondary structures. The blue outer ring segments indicate the novel is-ncRNAs, and the different branch colours indicate different types of known functional is-ncRNAs. The blue brackets indicate the eight potentially novel is-ncRNA classes, which are shown in a greater level of detail at the right side of the circular tree, and the green out ring indicates the tRNA cluster, which is shown in a greater level of detail at the left side of the circular tree. (B) Predicted secondary structures of the eight potentially novel is-ncRNA classes in P. falciparum. (C) Internal motifs (IM1 and IM2) of the novel intergenic is-ncRNAs beyond the eight novel classes.
Figure 4.
Experimental validation of novel is-ncRNAs.
(A) RT-PCR confirmation of the novel is-ncRNAs. Twelve of the is-ncRNAs are shown as examples, and the remaining 48 are documented in Figure S1 in file S2. Each is-ncRNA is shown in three adjacent lanes, from left to right: DNase-treated RNA RT-PCR (“+”), RT-PCR with no RNA template (left, “−”, negative control), and RT without reverse transcriptase (right, “−”, negative control). “M” indicates the 50 bp DNA ladder. “Length” indicates the expected size of the is-ncRNAs based on the Illumina/Solexa paired-end sequencing assembly data. (B) Northern blotting validation of six novel is-ncRNAs. U5 snRNA was used as an internal control. The arrows indicate the sizes of the is-ncRNAs; the black arrows indicate products with lengths consistent with those of the RT-PCR products, and the grey arrows indicate products with lengths that were larger than those determined by RT-PCR. “Length” indicates the expected size of the is-ncRNA based on the Illumina/Solexa paired-end sequencing assembly data and RT-PCR.
Figure 5.
Expression profiles analysis of the novel is-ncRNAs.
(A) qRT-PCR analysis of the expression of 13 novel is-ncRNAs during the intraerythrocytic developmental stage. U6 snRNA was used as an internal control to calculate the relative expression level (2−ΔΔCt) of each novel is-ncRNA. The known ncRNA U1 snRNA was used as reference controls. Statistically significant differences were determined using paired Student's T-test. **denotes p<0.01 and ***denotes p<0.001. (B) Relative expression analysis of the four novel antisense RNAs (blue) and their cis-encoded sense RNAs (red) during the intraerythrocytic developmental stages as determined by qRT-PCR. β-actin I was used as an internal control. The relative expression level was calculated as –ΔCt.
Table 2.
Details for the four antisense is-ncRNAs and their cis-encoded sense RNAs.