Abstract
The technique of ribosome profiling is based on the isolation of sequences around 30 nucleotides in size protected by mRNA-associated ribosomes, following digestion with specific nucleases, generating a footprint. After isolation and purification, these 30-nucleotide sequences are converted to a cDNA library and analyzed by deep sequencing, providing a high-precision picture of the translation process in vivo. In addition, this powerful technique allows for the study of several biological phenomena such as alternative splicing, alternative codon usage and initiation of translation by non-AUG codons. Furthermore, the ribosome footprinting technique has proved to be very efficient for studies of ribosome pause sites on mRNAs, which could act as key regulators in the translation process. Here we describe a modified protocol of the ribosome footprinting technique for translation efficiency analysis in Trypanosoma cruzi.
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References
Avila AR, Dallagiovanna B, Yamada-Ogatta SF, Monteiro-Goes V, Fragoso SP, Krieger MA, Goldenberg S (2003) Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis. Genet Mol Res 2(1):159–168. Review
Fragoso SP, Plazanet-Menut C, Carreira MA, Motta MC, Dallagiovana B, Krieger MA, Goldenberg S (2003) Cloning and characterization of a gene encoding a putative protein associated with U3 small nucleolar ribonucleoprotein in Trypanosoma cruzi. Mol Biochem Parasitol 126(1):113–117
Nardelli SC, Avila AR, Freund A, Motta MC, Manhães L, de Jesus TC, Schenkman S, Fragoso SP, Krieger MA, Goldenberg S, Dallagiovanna B (2007) Small subunit rRNA processome proteins are translationally regulated during differentiation of Trypanosoma cruzi. Eukaryot Cell 6(2):337–345
Holetz FB, Correa A, Ávila AR, Nakamura CV, Krieger MA, Goldenberg S (2007) Evidence of P-body-like structures in Trypanosoma cruzi. Biochem Biophys Res Commun 356:1062–1067
Holetz FB, Alves LR, Probst CM, Dallagiovanna B, Marchini FK, Manque P, Buck G, Krieger MA, Correa A, Goldenberg S (2010) Protein and mRNA content of TcDHH1-containing mRNPs in Trypanosoma cruzi. FEBS J 277:3415–3426
Costa JF, Ferrarini MG, Nardelli SC, Goldenberg S, Avila AR, Holetz FB (2018) Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery. Mem Inst Oswaldo Cruz 113:e1705317
Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218–223
Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147:789–802
Smircich P, Eastman G, Poubel S, Duhagon MA, Guerra-Slompo E, Garat B, Goldenberg S, Munroe DJ, Dallagiovanna B, Holetz F, Sotelo-Silveira JR (2015) Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi. BMC Genomics 16:443
Nilsen TW, Graveley BR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463(7280):457–463
Plotkin JB (2010) Transcriptional regulation is only half the story. Mol Syst Biol 6:406
Kolev NG, Franklin JB, Carmi S, Shi H, Michael S, Tschudi C (2010) The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution. PLoS Pathog 6(9):1001090
Touriol C, Bornes S, Bonnal S, Audigier S, Prats H, Prats AC, Vagner S (2003) Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons. Biol Cell 95(3–4):168–178
Vagner S, Galy B, Pyronnet S (2001) Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites. EMBO Rep 2(10):893–898
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Poubel, S.B., Holetz, F.B., Romagnoli, B.A.A., Goldenberg, S., Alves, L.R. (2020). Analysis of the In Vivo Translation Process in Trypanosoma cruzi Using Ribosome Profiling. In: Michels, P., Ginger, M., Zilberstein, D. (eds) Trypanosomatids. Methods in Molecular Biology, vol 2116. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0294-2_9
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DOI: https://doi.org/10.1007/978-1-0716-0294-2_9
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