Abstract:In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in translation. However, tRNAs can be returned to the nucleus by the evolutionarily conserved process called tRNA retrograde nuclear import, before relocalization back to the cytoplasm via a nuclear re-export step. Several important functions of these latter two trafficking events have been identified, yet the pathways are largely unknown. Therefore, we developed an ass… Show more
“…APB interacts with Q in tRNA, slowing its mobility in APB-gels (in addition to above, also see Kessler et al 2018 ; Cirzi and Tuorto 2021 ); in species in which Q is glycosylated, an adaptation can be incorporated ( Zhang et al 2020 ). A unique tRNA-specific approach was developed for northern blot detection of yW37 found on a single tRNA, the isoacceptor for Phe ( Nostramo and Hopper 2020 ). This exploits the specific reactivity of yW with aniline followed by hydrolysis with mild acid.…”
The ~22 mitochondrial and ~45 cytosolic tRNAs contain several dozen different posttranscriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression and their deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our lab developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ~10 years ago which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of an DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32 and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.
“…APB interacts with Q in tRNA, slowing its mobility in APB-gels (in addition to above, also see Kessler et al 2018 ; Cirzi and Tuorto 2021 ); in species in which Q is glycosylated, an adaptation can be incorporated ( Zhang et al 2020 ). A unique tRNA-specific approach was developed for northern blot detection of yW37 found on a single tRNA, the isoacceptor for Phe ( Nostramo and Hopper 2020 ). This exploits the specific reactivity of yW with aniline followed by hydrolysis with mild acid.…”
The ~22 mitochondrial and ~45 cytosolic tRNAs contain several dozen different posttranscriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression and their deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our lab developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ~10 years ago which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of an DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32 and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.
“…There is precedence for such differential requirements of shuttling proteins in the RNA world. The transfer RNA Phe (tRNA Phe ) molecules require Mex67/Mtr2, Xpo1 (CRM1), Los1 and Msn5 for the first round of nuclear export, while only requiring Mex67/Mtr2 and Xpo1 (CRM1), but neither Los1 or Msn5 for a subsequent second re-export (Nostramo and Hopper 2020 ). This agrees with another report showing that tRNA export proteins have differential preferences for various tRNAs, and that Mex67/Mtr2-Los1 is utilised by a subset of tRNAs only for the initial nuclear export (Chatterjee et al 2017 ).…”
Section: Additional Rounds Of Tlc1 Shuttlingmentioning
As the limiting component of the budding yeast telomerase, the Tlc1 RNA must undergo multiple consecutive modifications and rigorous quality checks throughout its lifecycle. These steps will ensure that only correctly processed and matured molecules are assembled into telomerase complexes that subsequently act at telomeres. The complex pathway of Tlc1 RNA maturation, involving 5'- and 3'-end processing, stabilisation and assembly with the protein subunits, requires at least one nucleo-cytoplasmic passage. Furthermore, it appears that the pathway is tightly coordinated with the association of various and changing proteins, including the export factor Xpo1, the Mex67/Mtr2 complex, the Kap122 importin, the Sm7 ring and possibly the CBC and TREX-1 complexes. Although many of these maturation processes also affect other RNA species, the Tlc1 RNA exploits them in a new combination and, therefore, ultimately follows its own and unique pathway. In this review, we highlight recent new insights in maturation and subcellular shuttling of the budding yeast telomerase RNA and discuss how these events may be fine-tuned by the biochemical characteristics of the varying processing and transport factors as well as the final telomerase components. Finally, we indicate outstanding questions that we feel are important to be addressed for a complete understanding of the telomerase RNA lifecycle and that could have implications for the human telomerase as well.
“…An overlapping and different role of Mex67 and Mtr2 was also suggested for budding yeast. This is however a different matter, considering that S. cerevisiae lacks the gene for Q-tRNA modification enzyme (Nostramo and Hopper, 2020). These data demonstrate the dynamic nature of tRNA trafficking depending on their modification status and vice versa .…”
Section: Introductionmentioning
confidence: 99%
“…1A). After intron removal, tRNA travels back to the nucleus with the help of the protein Mtr10 to get m 1 G37 and finally is re-exported to the cytoplasm where the remaining four enzymes (Tyw1-4) for wybutosine biosynthesis reside (Ohira and Suzuki, 2011; Nostramo and Hopper, 2020). Fig.…”
Section: Introductionmentioning
confidence: 99%
“…In the final step, both Mex67 and Crm1 mediate the constitutive re-export of tRNA Phe to the cytoplasm, where wybutosine (yW) (in yellow) is added to m 1 G in a sequential series of reactions by Tyw1-4. Notably, the canonical exporters Los1 and Msn5 are dispensable in this transport step (Chatterjee et al ., 2018; Hopper and Nostramo, 2019; Nostramo and Hopper, 2020). (B) A model for subcellular trafficking and maturation of the tyrosyl-tRNA (tRNA Tyr ) in T. brucei .…”
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