Inosine modifications in human tRNAs are incorporated at the precursor tRNA level.
Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
Adrian Gabriel
AG
Torres
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
David
D
Piñeyro
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
Marta
M
Rodríguez-Escribà
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
Noelia
N
Camacho
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
Oscar
O
Reina
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
Adélaïde
A
Saint-Léger
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain.
Liudmila
L
Filonava
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain Catalan Institution for Research and Advanced Studies (ICREA), P/Lluis Companys 23, Barcelona, 08010 Catalonia, Spain.
Eduard
E
Batlle
Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona, 08028 Catalonia, Spain Catalan Institution for Research and Advanced Studies (ICREA), P/Lluis Companys 23, Barcelona, 08010 Catalonia, Spain lluis.ribas@irbbarcelona.org.
Lluís
L
Ribas de Pouplana
D000243:Adenosine Deaminase / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007288:Inosine / Q000378:metabolism*; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA
D012322:RNA Precursors; D007288:Inosine; D012343:RNA, Transfer; C000621957:ADAT2 protein, human; D000243:Adenosine Deaminase