Seminarium Instytutowe

Dr Abu Musa Md Talimur Reza

Laboratory of RNA Metabolism in Immune Responses

Investigation of the effects of tRNA genes knock-out in human cells


In mammalian cells, there are multiple copies of tRNA genes within the same tRNA isotype or isoacceptor
families. Curiously, not all of these tRNA genes are equally transcribed, and many of them being completely
silent. The full understanding of how individual tRNA genes are regulated is still missing. Among a few
hypotheses that were posed is the influence of chromatin context, the existence of proximal Pol II
transcriptional unit, and/or the strength of the terminator downstream of a tRNA gene body. Moreover, it is
not known whether the expression of the tRNAs is coordinated as a group, at the isoacceptor family level, or,
in other words, whether the cells activate low or non-expressed tRNA genes when the active fail to sustain
the required cellular needs.
The aim of this work is to test whether: 1) downregulation of active tRNA genes within isoacceptor family
leads to activation of the silent ones; 2) genomic manipulation of the terminator sequence affects tRNA gene
activity in vivo; 3) translocation of silent tRNA into the active chromatin context activates its transcription?
In order to address these questions we employed CRISPR/Cas9-based approach to completely remove
individual tRNA genes within tRNATrp(CCA) family or to manipulate downstream terminator sequences
directly in the genome of human cancer cells. We also used retroviral transduction to transfer silent tRNA
gene into the open chromatin context.
We were able to knock-out four active tRNATrp(CCA) genes, leaving only one active and two inactive genes.
We could not observe any changes in Pol III occupancy at the remaining genes, suggesting no coordinate
regulation of these tRNA genes. Strikingly, only one active tRNATrp(CCA) gene is enough to maintain normal
biological functions of human cells.
Furthermore, genetic manipulation of the terminator sequence (removal or introducing) showed no impact
on Pol III binding at the tRNA genes. Finally, we did not observe activation of silent tRNATrp(CCA) after its
translocation into open chromatin context. These data suggest the other mechanism(s) control activity of
tRNA genes in vivo.



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