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Published on: January 13, 2014
Interrogation of RNA-bound proteome with XRNAX illuminates molecular alterations in the mouse brain affected with
Marta Sztachera1, Weronika Wendlandt-Stanek1, Remigiusz A Serwa2
1Department of Non-coding RNAs, Institute of Bioorganic Chemistry of the Polish Academy of Sciences, 61-704 Poznan, Poland.
Abstract:
RNA-protein interactions orchestrate hundreds of pathways in homeostatic and stressed cells. We applied an RNA-protein interactome capture method called protein cross-linked RNA extraction (XRNAX) to shed light on the RNA-bound proteome in dysmyelination. We found sets of canonical RNA-binding proteins (RBPs) regulating alternative splicing and engaged in the cytoplasmic granules to be perturbed at the level of their RNA interactome. We validated these observations for PCBP1 and MBNL1. We show that the number of PCBP1 bodies is markedly increased in the mossy cells of the hippocampus and that the pattern of MBNL1-regulated alternatively spliced exons differs between the myelin-deficient and the wild-type brain, which is likely associated with Mbnl1 splicing perturbation and circular RNA generation from this locus. In the broader perspective, our results demonstrate that, with the application of the RNA-protein interactome approach, we can uncover alterations in RBP functioning in the disease context that are not always directly visible from their mRNA or protein levels.
Insights
This study reveals altered RNA-protein interactions in dysmyelination using the XRNAX method. Key RNA-binding proteins (RBPs) show changed RNA interactomes, impacting alternative splicing and cellular granules.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- RNA-protein interactions are crucial for cellular functions, including alternative splicing and stress responses.
- Dysmyelination involves disruptions in these interactions, but the specific changes in the RNA-bound proteome are not fully understood.
- Traditional methods may not capture subtle alterations in RNA-binding protein (RBP) function during disease.
Purpose of the Study:
- To investigate the RNA-bound proteome in dysmyelination using a novel interactome capture method.
- To identify specific RNA-binding proteins (RBPs) and their RNA interactions that are perturbed in this condition.
- To understand the functional consequences of these perturbations on cellular processes like alternative splicing.
Main Methods:
- Application of protein cross-linked RNA extraction (XRNAX) to capture RNA-protein complexes.
- Analysis of the RNA interactome of RBPs in myelin-deficient versus wild-type brain tissue.
- Validation of findings for specific RBPs (PCBP1 and MBNL1) using cellular and molecular techniques.
Main Results:
- Perturbations were observed in canonical RBPs involved in alternative splicing and cytoplasmic granules.
- The number of PCBP1 bodies increased in hippocampal mossy cells.
- MBNL1-regulated alternative splicing patterns differed in myelin-deficient brains, linked to splicing changes and circular RNA generation.
Conclusions:
- The RNA-protein interactome approach effectively uncovers RBP functional alterations in disease, even when mRNA or protein levels are unchanged.
- Dysmyelination is associated with significant changes in RNA-binding protein interactions and their regulatory roles.
- These findings highlight the importance of studying RNA-protein interactions to understand disease mechanisms.
