ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels
Abstract
:1. Introduction
2. Results
2.1. mRNAs Bound by ThrRS
2.2. RPC10 Is Bound by ThrRS through an Element Predicted to Form Cognate tRNA ASL
2.3. Altered ThrRS Binding upon Elimination of the ASL-like Element
2.4. The Predicted ASL-Mimic Is Important for RPC10 Translation
2.5. Mutations within the Predicted ASL-like Site Lead to an Impact on tRNAThr Expression
3. Discussion
3.1. The Importance of the Predicted ASL-like for Translation Regulation of RPC10
3.2. Regulation of Expression by tRNA Mimicry
3.3. Regulation of tRNA Expression by ThrRS
4. Materials and Methods
4.1. Yeast Strains, Growth Conditions, and Plasmids
4.2. RBP ImmunoPrecipitation (RIP) and RNA-seq Analyses
4.3. CRISPR/Cas9-Mediated Recombination
4.4. Polysome Analysis
4.5. tRNA Quantification by RT-qPCR
4.6. Protein Co-Immunoprecipitation
4.7. Mass Spectrometry (MS/MS) Analysis
Lab Code | Strain Name | Description | Genotype | Source |
---|---|---|---|---|
YA1 | BY4741 | Wild type | Mat a, his3∆1, leu2∆0, met15∆0,ura3∆0 | [39] |
YA1545 | THS1-GFP | THS1 (YIL078W) C-terminally tagged with GFP, in BY4741 | MATa, leu2Δ0, met15Δ0, ura3Δ0, THS1: GFP (S65T)-HIS3 | [40] |
YA1595 | THS1-GFP RPC10-6M | THS1 (YIL078W) C-terminally tagged with GFP, BY4741. RPC10 gene was mutated (AGTCCGTTGTAAGGAC to GGTGCGGTGCAAAGAT) | MATa, leu2Δ0, met15Δ0, ura3Δ0, THS1: GFP (S65T)-HIS3, rpc10-6m | This study |
YA1619 | GFP-RPC10 | N terminally GFP-tagged RPC10 (YHR143W-A), BY4741 | MATa, leu2Δ0, met15Δ0, ura3Δ0, GFP(S65T)-HIS3: RPC10 | [41] |
YA1620 | GFP-RPC10 6M | N terminally GFP-tagged RPC10 (YHR143W-A), BY4741. RPC10 gene was mutated (AGTCCGTTGTAAGGAC to GGTGCGGTGCAAAGAT) | MATa, leu2Δ0, met15Δ0, ura3Δ0, GFP(S65T)-HIS3: RPC10 | This study |
Lab Code | Plasmid Name | Description | Source |
---|---|---|---|
pA1067 | bRA66 | (Empty Backbone) GAL1 driven Cas9 Addgene #100952 | [42] |
pA1194 | RPC10 + 143 gRNA | bRA66 with RPC10 +143 (AACTGATGCAGTCCGTTGTA) targeting gRNA cloned into BplI sites | This study |
gRNA Oligos | |
Primer Name | Primer sequence |
RPC10 gRNA-143 F | AACTGATGCAGTCCGTTGTAGTTTT |
RPC10 gRNA-143 R | TACAACGGACTGCATCAGTTGATCA |
Donor DNA Oligos | |
Primer Name | Primer sequence |
RPC10 Random Donor DNA F | TAGTAAATTATCTTTATCCAGAACTGATGCNGTNCGNTGYAARGAYTGTGGTCATAGAATCCTGTTGAAGGCTAGGACTA |
RPC10 Random Donor DNA R | TAGTCCTAGCCTTCAACAGGATTCTATGACCACARTCYTTRCANCGNACNGCATCAGTTCTGGATAAAGATAATTTACTA |
RPC10 6M Donor DNA F | TAGTAAATTATCTTTATCCAGAACTGATGCgGTgCGgTGcAAaGAtTGTGGTCATAGAATCCTGTTGAAGGCTAGGACTA |
RPC10 6M Donor DNA R | TAGTCCTAGCCTTCAACAGGATTCTATGACCACAaTCtTTgCAcCGcACcGCATCAGTTCTGGATAAAGATAATTTACTA |
qPCR Oligos | |
Primer Name | Primer sequence |
RPC10 seq F | ATGTCTCGCGAAGGGTTC |
RPC10 seq R | TCAAATTGAACCAATCTCTTAGTCC |
RT-qPCR Oligos | |
Primer Name | Primer sequence |
RPC10 F | CGCGAAGGGTTCCAGATT |
RPC10 R | CTTCAAAGTTGCCGTTCTAGC |
Act1 F | GATTCTGAGGTTGCTGCTTTG |
Act1 R | ACCGACGATAGATGGGAAGA |
tRNA-Thr-AGT F | CAAGTTGGTAAGGCGCCAC |
tRNA-Thr-AGT R | TCGGATTTGAACCGATGATCTCC |
tRNA-Leu-CAA F | TTTGGCCGAGCGGTCTAAGG |
tRNA-Leu-CAA R | TGCATCTTACGATACCTGAGCTT |
5S rRNA F | GCGGCCATATCTACCAGAAA |
5S rRNA R | GTATGGTCACCCACTACACTAC |
Northern probes | |
tRNAThr biotin | /5BiosG/GGATTTGAACCGATGATCT |
5S biotin | /5BiosG/TGGTAGATATGGCCGCAACC |
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Levi, O.; Mallik, M.; Arava, Y.S. ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels. Genes 2023, 14, 462. https://doi.org/10.3390/genes14020462
Levi O, Mallik M, Arava YS. ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels. Genes. 2023; 14(2):462. https://doi.org/10.3390/genes14020462
Chicago/Turabian StyleLevi, Ofri, Monalisha Mallik, and Yoav S. Arava. 2023. "ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels" Genes 14, no. 2: 462. https://doi.org/10.3390/genes14020462
APA StyleLevi, O., Mallik, M., & Arava, Y. S. (2023). ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels. Genes, 14(2), 462. https://doi.org/10.3390/genes14020462