Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

Abstract

Background: Small interfering RNA (siRNA) molecules mediate sequence specific silencing in RNA interference (RNAi), a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA) molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories.

Results: Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24-26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C.

Conclusion: These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

Figure 1

Length distribution of siRNAs. The siRNAs were obtained by high-throughput sequencing of mixed stage C. elegans populations from two library sources (Lee et al., 2006; Ruby et al., 2006). The height of the bars indicates the number of siRNAs for the respective length. The shaded portion of the histograms indicates the number of siRNAs with an mRNA sequence match. A) Length distribution of all 7136 available short RNA sequences from C. elegans available from the two library sources. The length of sequences ranged from 12- to 37 nucleotides. A total of 4024 siRNA sequences matched with an mRNA sequence from the two library sources. B) Length distribution of siRNAs from the library reported in Lee et al., 2006. C) Length distribution of siRNAs from the library reported in Ruby et al., 2006.

Figure 2

Length distribution of the multi-read siRNAs. Multi-read siRNAs were obtained from the library in Ruby et al., 2006 as described in the methods section. The lengths were calculated and the number of counts for each nucleotide length was plotted.

Figure 3

Length distribution and target sites of siRNAs complementary to Argonaute-related mRNA NM_075351 (F55C9.3). The siRNA identifier, number of reads, length, siRNA sequence, and position along the transcript is shown. The short bars indicate the position along the transcript and the numbers indicate the nucleotide position along the sequence. There are one to two nucleotide shorter versions of certain siRNAs that have been read multiple times. The number of reads was not available (n.a.) in some cases because those siRNAs were obtained from Lee et al. (2006).

Figure 4

The distribution of 5' nucleotides in siRNAs grouped by the length. Each bar represents the percentage of siRNAs for the given length. The shading indicates the 5' starting nucleotide.

Figure 5

The frequency sequence graphs (logos) of siRNAs which have putative target mRNAs. siRNA sequences were collected and divided by size as described in methods. Sequence logos were generated using WebLogo . The siRNA length is shown on the left and the number of siRNAs in each length category is shown on the right.