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. Here, 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 a 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 that show 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.

1 salivary-gland sporozoites point to tight regulatory control 2 and potential mechanisms for liver-stage differentiation. ABSTRACT 2 8Plasmodium vivax is the key obstacle to malaria elimination in Asia and Latin America, 2 9 largely attributed to its ability to form resilient hypnozoites (sleeper-cells) in the host liver 3 0 that escape treatment and cause relapsing infections. The decision to form hypnozoites is 3 1 made early in the liver infection and may already be set in sporozoites prior to invasion. To 3 2 better understand these early stages of infection, we undertook a comprehensive 3 3 transcriptomic and histone epigenetic characterization of P. vivax sporozoites. The salivary-3 4 gland sporozoite transcriptome is heavily composed of transcripts associated with functions 3 5 needed for early infection of the vertebrate host and development within hepatocytes. 3 6Through comparisons to recently published proteome data for the P. vivax sporozoite, our 3 7 study finds that although highly transcribed, these transcripts are not detectable as proteins 3 8 and may be regulated through translational repression; a finding we test for a small subset of 3 9 transcripts and proteins through immunofluorescent microscopy of sporozoites and liver 4 0 stages in humanized mice. We identify differential transcription between the sporozoite and 4 1 published transcriptomes of asexual blood-stages and mixed versus hypnozoite-enriched liver 4 2 stages. These comparisons point to multiple layers of transcriptional, post-transcriptional and 4 3 post-translational control that appear active in sporozoites and to a lesser extent hypnozoites, 4 4 but largely absent in replicating liver schizonts or mixed blood-stages. Common transcripts 4 5 up-regulated in sporozoites and hypnozoites compared to mixed (i.e., schizont) liver-stages 4 6identify genes linked to dormancy/persistence in bacteria, amoebae and plants. We also 4 7 characterise histone epigenetic modifications in the P. vivax sporozoite and explore their role 4 8 in regulating transcription. Collectively, these data support the hypothesis that the sporozoite 4 9 as a tightly programmed stage primed to infect the human host and identifies potential 5 0 mechanisms for hypnozoite-formation that may be further explored in liver stage models. 5 1 5 2