Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia

Abstract

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3′ UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3′ end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Comprehensive characterization of Cysteine-rich protein-coding genes of Giardia lamblia and their role during antigenic variation

“…Moreover, these PAS were found to be depleted in coding regions while occasionally overlap with stop codons, like in the murine parasite G. muris [57]. The 3' UTR lengths generated by that approach had a median of 59 nt, which is consistent with the idea that 3' UTRs of G. lamblia are unusually short [47,56]. Then, the presence of PAS in the 3 ′ end of all Cys-rich genes was determined showing that all 136 VSPs have an extended PAS with the sequence ACUUAGRUAGURAAYRY (R = purines and Y = pyrimidines) (Fig.…”

“…For previously annotated HCMPs, the length of the 3' UTR has been determined to be variable, ranging from only a few nt up to thousands [47,56]. Consistent with this finding, only 67 of 94 CRMPs have AGURAA in the 2000 nt downstream of the stop codon.…”

“…It was previously stated that the poly(A) signal sites (PAS) of Giardia genes was AGRAAA, where R is a purine [47,53]. Recently, it was verified on a genome-wide scale that G. lamblia uses an AGURAA PAS [56]. AGUAAA and AGUGAA were present in 45% and 15% of protein coding genes, respectively.…”

Spliceosomal introns in the diplomonad parasite Giardia duodenalis revisited

“…While our study focused on short-read RNA-seq of expressed transcripts to identify splice junctions in Giardia ’s transcriptome, other multi-omics datasets are now available for additional study in the future to validate and polish the SJs we report here and, more broadly, to refine bioinformatics strategies for annotating microbial eukaryote genomes (including intron discovery) [ 42 ]. The inclusion of long-read transcriptome sequencing can help further refine the identified SJs due to increased alignment specificity to the flanking exonic regions, and can also be useful for future searches for additional trans- splicing junctions [ 43 ]. Similarly, MS-based proteome sequencing may also provide additional lines of evidence to support or refute the existence of introns in genes denoted as ‘functional’ in our study (and also trans -spliced genes) as well as address the possibility that some introns may also have secondary functions as stable RNAs.…”