Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo
Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fai...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2024-12-01
|
| Series: | RNA Biology |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2019.1657788 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849220680559624192 |
|---|---|
| author | Gonzalo I. Mendoza-Ochoa J. David Barrass Isabella E. Maudlin Jean D. Beggs |
| author_facet | Gonzalo I. Mendoza-Ochoa J. David Barrass Isabella E. Maudlin Jean D. Beggs |
| author_sort | Gonzalo I. Mendoza-Ochoa |
| collection | DOAJ |
| description | Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this ‘feed-back’ effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions. |
| format | Article |
| id | doaj-art-fe41cc9df50f45d7853c0dfdf966cbe4 |
| institution | Kabale University |
| issn | 1547-6286 1555-8584 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj-art-fe41cc9df50f45d7853c0dfdf966cbe42024-12-05T12:03:26ZengTaylor & Francis GroupRNA Biology1547-62861555-85842024-12-0116121775178410.1080/15476286.2019.1657788Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivoGonzalo I. Mendoza-Ochoa0J. David Barrass1Isabella E. Maudlin2Jean D. Beggs3Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UKWellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UKWellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UKWellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UKPre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this ‘feed-back’ effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.https://www.tandfonline.com/doi/10.1080/15476286.2019.1657788Auxinpre-mRNA splicingPrp22protein depletionyeast |
| spellingShingle | Gonzalo I. Mendoza-Ochoa J. David Barrass Isabella E. Maudlin Jean D. Beggs Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo RNA Biology Auxin pre-mRNA splicing Prp22 protein depletion yeast |
| title | Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo |
| title_full | Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo |
| title_fullStr | Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo |
| title_full_unstemmed | Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo |
| title_short | Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo |
| title_sort | blocking late stages of splicing quickly limits pre spliceosome assembly in vivo |
| topic | Auxin pre-mRNA splicing Prp22 protein depletion yeast |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2019.1657788 |
| work_keys_str_mv | AT gonzaloimendozaochoa blockinglatestagesofsplicingquicklylimitsprespliceosomeassemblyinvivo AT jdavidbarrass blockinglatestagesofsplicingquicklylimitsprespliceosomeassemblyinvivo AT isabellaemaudlin blockinglatestagesofsplicingquicklylimitsprespliceosomeassemblyinvivo AT jeandbeggs blockinglatestagesofsplicingquicklylimitsprespliceosomeassemblyinvivo |