RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein
A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from <i>Saccharomyces cerevisiae</i> lacks sequence homology with retroviral Gag, but is functionally related. In a...
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MDPI AG
2021-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/16/9103 |
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| author | Julita Gumna Angelika Andrzejewska-Romanowska David J. Garfinkel Katarzyna Pachulska-Wieczorek |
| author_facet | Julita Gumna Angelika Andrzejewska-Romanowska David J. Garfinkel Katarzyna Pachulska-Wieczorek |
| author_sort | Julita Gumna |
| collection | DOAJ |
| description | A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from <i>Saccharomyces cerevisiae</i> lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo. |
| first_indexed | 2024-03-10T08:44:16Z |
| format | Article |
| id | doaj.art-c3b8a7fca64040ef859aa0da0180fead |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T08:44:16Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-c3b8a7fca64040ef859aa0da0180fead2023-11-22T08:05:33ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-08-012216910310.3390/ijms22169103RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag ProteinJulita Gumna0Angelika Andrzejewska-Romanowska1David J. Garfinkel2Katarzyna Pachulska-Wieczorek3Department of Structure and Function of Retrotransposons, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandDepartment of Structure and Function of Retrotransposons, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USADepartment of Structure and Function of Retrotransposons, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandA universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from <i>Saccharomyces cerevisiae</i> lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.https://www.mdpi.com/1422-0067/22/16/9103Ty1 retrotransposonGagprotein-RNA interactionsbinding affinityRNA packagingMST |
| spellingShingle | Julita Gumna Angelika Andrzejewska-Romanowska David J. Garfinkel Katarzyna Pachulska-Wieczorek RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein International Journal of Molecular Sciences Ty1 retrotransposon Gag protein-RNA interactions binding affinity RNA packaging MST |
| title | RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein |
| title_full | RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein |
| title_fullStr | RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein |
| title_full_unstemmed | RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein |
| title_short | RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein |
| title_sort | rna binding properties of the ty1 ltr retrotransposon gag protein |
| topic | Ty1 retrotransposon Gag protein-RNA interactions binding affinity RNA packaging MST |
| url | https://www.mdpi.com/1422-0067/22/16/9103 |
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