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A topogenic role for the oncogenic N-terminus of TLS: nucleolar localization when transcription is inhibited
H Zinszner1, D Immanuel, Y Yin
1Department of Medicine, Skirball Institute of Biomolecular Medicine, NYU Medical Center, New York, NY 10016, USA.
Abstract:
TLS (FUS) and the related gene EWS encode the N-terminal portion of many fusion oncoproteins involved in human sarcomas and leukemia. TLS is an RNA-binding nuclear protein that is identical to hnRNP P2 and may be implicated in mRNA metabolism. When RNA polymerase II is inhibited, TLS immunostaining in the nucleus is dramatically altered, from its normal diffuse nucleoplasmic pattern to accumulation in dense nuclease-resistant aggregates. Co-immunostaining with antibodies to fibrillarin or p80 coilin and immunoelectron microscopy revealed that the TLS aggregates are associated with the nucleolus and are distinct from other known structures such as the coiled body or the interchromatin granule. Injection of cells with an oligodeoxynucleotide that disrupts splicing does not result in redistribution of TLS, indicating that the event is specific to inhibition of transcription. Oncoproteins that contain the N-terminal domain from either TLS, EWS or their Drosophila homologue, SARFH (CAZ), are also targeted to the same structure. These findings suggest a correlation between the topogenic and transforming activities of TLS and EWS N-termini and imply the existence of cellular targets that are shared by the germ-line encoded proteins and their oncogenic derivatives.
Insights
The study reveals that inhibiting transcription causes the TLS protein to form aggregates in the nucleolus, a structure distinct from known nuclear bodies. These findings link TLS protein aggregation to its oncogenic potential in sarcomas and leukemia.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The TLS (FUS) and EWS genes encode N-terminal domains of fusion oncoproteins implicated in human sarcomas and leukemia.
- TLS is an RNA-binding nuclear protein involved in mRNA metabolism, with a diffuse nucleoplasmic localization under normal conditions.
Purpose of the Study:
- To investigate the cellular localization and behavior of the TLS protein upon inhibition of transcription.
- To determine the nature of TLS aggregates and their relationship to known nuclear structures.
- To explore whether oncogenic fusion proteins containing TLS or EWS N-termini share cellular targets.
Main Methods:
- Immunostaining to visualize TLS protein localization in the nucleus.
- Co-immunostaining with antibodies against fibrillarin and p80 coilin.
- Immunoelectron microscopy to identify aggregate structures.
- Cellular assays involving inhibition of RNA polymerase II and splicing disruption.
Main Results:
- Inhibition of RNA polymerase II, but not splicing, caused TLS to shift from a diffuse nuclear pattern to dense, nuclease-resistant aggregates.
- These TLS aggregates were found to be associated with the nucleolus and distinct from coiled bodies or interchromatin granules.
- Oncoproteins containing TLS or EWS N-termini were also localized to these nucleolar-associated structures.
Conclusions:
- TLS protein aggregation is specifically linked to transcription inhibition and occurs in nucleolar-associated structures.
- The findings suggest a correlation between the targeting of TLS/EWS N-termini to these structures and their oncogenic activities.
- Cellular targets may be shared by germ-line TLS/EWS proteins and their oncogenic derivatives.