The transcriptome of Leishmania major in the axenic promastigote stage: transcript annotation and relative expression levels by RNA-seq

Abstract

Background: Although the genome sequence of the protozoan parasite Leishmania major was determined several years ago, the knowledge of its transcriptome was incomplete, both regarding the real number of genes and their primary structure.

Results: Here, we describe the first comprehensive transcriptome analysis of a parasite from the genus Leishmania. Using high-throughput RNA sequencing (RNA-seq), a total of 10285 transcripts were identified, of which 1884 were considered novel, as they did not match previously annotated genes. In addition, our data indicate that current annotations should be modified for many of the genes. The detailed analysis of the transcript processing sites revealed extensive heterogeneity in the spliced leader (SL) and polyadenylation addition sites. As a result, around 50% of the genes presented multiple transcripts differing in the length of the UTRs, sometimes in the order of hundreds of nucleotides. This transcript heterogeneity could provide an additional source for regulation as the different sizes of UTRs could modify RNA stability and/or influence the efficiency of RNA translation. In addition, for the first time for the Leishmania major promastigote stage, we are providing relative expression transcript levels.

Conclusions: This study provides a concise view of the global transcriptome of the L. major promastigote stage, providing the basis for future comparative analysis with other development stages or other Leishmania species.

Figure 1

Transcript assembling and annotation from RNASeq data. The figure shows a region of the chromosome 7. Panel A: reads aligned in this region; a small window of the total mapped reads is shown (bottom panel); the relative coverage (logarithmic scale) is depicted as a sky-line on the panel. Each vertical dash represents a read. Reads aligned with the plus strand of the chromosome are shown in pink and those aligned with the minus strand in violet (note that the direction of the reads was assigned arbitrarily, as sequencing was not oriented). Panel B: mapping of SL-containing reads. Panel C: previously annotated L. major genome (GeneDB database). Panel D: crude transcripts as assembled by Cufflinks. Panel E: new transcript annotation after mapping of both SL addition sites and the 3’ ends generated by polyadenylation. The images were generated after loading the RNA-seq data in the Integrative Genomics Viewer (IGV 2.1) [23].

Figure 2

Mis-annotation of LmjF04.0860 gene. (A) Upper panels show the mapping of RNA-seq reads (either total or SL-containing reads) in the genomic region containing the annotated LmjF04.0860 gene; the bottom panel contains the transcripts delimited in this region. Arrows indicate SL addition sites, the red arrow points at the main SL addition site. Reads aligned with the plus strand of the chromosome are shown in violet and those aligned with the minus strand in pink (note that the direction of the reads was a consequence of sequencing process, as sequencing was not oriented). (B) Nucleotide sequence (and predicted amino acid sequence) of the LmjF04.0860 gene as annotated in the GeneDB database [25]. Shaded in gray is shown the position of the main AG dinucleotide used for trans-splicing in the LmjF.04.T0860 transcripts, and underlined are those AG dinucleotide representing alternative SL addition sites. Shaded in green it is shown the first ATG found downstream the SL addition sites. (C) Alignment between the protein predicted in the LmjF.04.T0860 transcript and the Tb927.9.8290 protein annotated in the T. brucei GeneDB database [25]. Identical amino acids are shaded in gray.

Figure 3

Relative expression levels of transcripts derived from loci coding for HSP70 and ribosomal protein L23. Panel A: upper, current annotation of the two types of genes found in the L. major HSP70 locus, LmjF28.2770 (also known as HSP70-II) and LmjF28.2780 (HSP70-I); bottom, transcript annotation, as defined in this study, for the L. major HSP70 locus. Panel B: upper, current annotation of the two genes coding for the ribosomal protein L23, LmjF35.3790 and LmjF35.3800; bottom, transcripts annotated in this study. Each locus is composed by two types of genes with identical ORFs (blue boxes). Transcript mapping has allowed the identification of 5’-UTRs (purple boxes) and 3’-UTRs (green boxes). The number of reads mapped by Cufflinks within the different regions is shown at bottom.

Figure 4

Nucleotide frequencies for sequences surrounding SL and polyadenylation addition sites. Panels show the compositional profiles of sequences around the main SL addition sites (n = 9530), alternative SL addition sites (n = 4531), main polyadenylation sites (n = 3178) and alternative polyadenylation sites (n = 1238).