Eukaryotic transfer RNAs (tRNAs) are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function for protein synthesis. The evolutionarily conserved β-importin family member Los1 (Exportin-t) has been the only exporter known to execute nuclear export of newly transcribed intron-containing pre-tRNAs. Interestingly, is unessential in all tested organisms. As tRNA nuclear export is essential, we previously interrogated the budding yeast proteome to identify candidates that function in tRNA nuclear export. Here, we provide molecular, genetic, cytological, and biochemical evidence that the Mex67-Mtr2 (TAP-p15) heterodimer, best characterized for its essential role in mRNA nuclear export, cofunctions with Los1 in tRNA nuclear export. Inactivation of Mex67 or Mtr2 leads to rapid accumulation of end-matured unspliced tRNAs in the nucleus. Remarkably, merely fivefold overexpression of Mex67-Mtr2 can substitute for Los1 inΔ cells. Moreover, in vivo coimmunoprecipitation assays with tagged Mex67 document that the Mex67 binds tRNAs. Our data also show that tRNA exporters surprisingly exhibit differential tRNA substrate preferences. The existence of multiple tRNA exporters, each with different tRNA preferences, may indicate that the proteome can be regulated by tRNA nuclear export. Thus, our data show that Mex67-Mtr2 functions in primary nuclear export for a subset of yeast tRNAs.
SUMMARY
Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease
in vitro
. We apply CRISPR/Cas9 genome editing technology to introduce three
RBM20
mutations in iPSCs and differentiate them into iPSC-CMs to establish an
in vitro
model of RBM20 mutant
dilated cardiomyopathy
(DCM). In iPSC-CMs harboring a known causal
RBM20
variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-
trans
retinoic acid upregulates
RBM20
expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal
RBM20
mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in
RBM20
.
In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although~600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 59 termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 59-to-39 exonuclease Xrn1, which has specificity for RNAs with 59 monophosphate.
tRNA is essential for translation and decoding of the proteome. The yeast proteome responds to stress and tRNA biosynthesis contributes in this response by repression of tRNA transcription and alterations of tRNA modification. Here we report that the stress response also involves processing of pre-tRNA 3 ′ termini. By a combination of Northern analyses and RNA sequencing, we show that upon shift to elevated temperatures and/or to glycerol-containing medium, aberrant pre-tRNAs accumulate in yeast cells. For pre-tRNA UAU Ile and pre-tRNA UUU Lys these aberrant forms are unprocessed at the 5 ′ ends, but they possess extended 3 ′ termini. Sequencing analyses showed that partial 3 ′ processing precedes 5 ′ processing for pre-tRNA UAU Ile . An aberrant pre-tRNA Tyr that accumulates also possesses extended 3 ′ termini, but it is processed at the 5 ′ terminus. Similar forms of these aberrant pretRNAs are detected in the rex1Δ strain that is defective in 3 ′ exonucleolytic trimming of pre-tRNAs but are absent in the lhp1Δ mutant lacking 3 ′ end protection. We further show direct correlation between the inhibition of 3 ′ end processing rate and the stringency of growth conditions. Moreover, under stress conditions Rex1 nuclease seems to be limiting for 3 ′ end processing, by decreased availability linked to increased protection by Lhp1. Thus, our data document complex 3 ′ processing that is inhibited by stress in a tRNA-type and condition-specific manner. This stress-responsive tRNA 3 ′ end maturation process presumably contributes to fine-tune the levels of functional tRNA in budding yeast in response to environmental conditions.
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