Abstract
Interactions of long non-coding RNAs (lncRNA) with proteins play important roles in the regulation of many cellular processes. PANDAR (Promotor of CDKN1AAntisense DNA damage Activated RNA) is a lncRNA that is transcribed in a p53-dependent manner from the CDKN1A promoter and is involved in the regulation of proliferation and senescence. Overexpression of PANDAR has been observed in several tumor species and correlated with a poor prognosis for patient survival rate. Depending on the cellular state, PANDAR is known to interact with proteins such as the nuclear transcription factor Y subunit A (NF-YA) and the scaffold attachment factor A (SAF-A). However, a comprehensive analysis of the PANDAR interactome was missing so far. Therefore, we applied peptide nucleic acid (PNA)-based pull-downs combined with quantitative mass spectrometry to identify new protein binding partners. We confirmed potential candidates like U2AF65 and PTBP1, known to be involved in RNA processing. Furthermore, we observed that overexpression of PANDAR leads to a reduced level of the short pro-apoptotic BCL-X splice variant (BCL-XS) which is regulated by PTBP1. Simultaneous overexpression of PTBP1 was able to rescue this effect. Overall, our data suggest a role for PANDAR in the regulation of splicing events via its interaction partner PTBP1.
Highlights
Non-coding RNAs regulate different cellular mechanisms by interacting with all kind of biomolecules, such as other nucleic acids, proteins or small molecules and co-factors, thereby modifying their subsequent functions[1,2,3]
To identify PANDAR bound proteins, we used the combination of a peptide nucleic acid (PNA)-based PANDAR pull-down followed by quantitative mass spectrometry after stable isotope labeling in cell culture (SILAC)
By use of the PNA-based PANDAR pull-down followed by quantitative mass spectrometry after SILAC, we were able to determine 22 potential binding candidates of the PANDAR interactome
Summary
Non-coding RNAs (ncRNAs) regulate different cellular mechanisms by interacting with all kind of biomolecules, such as other nucleic acids, proteins or small molecules and co-factors, thereby modifying their subsequent functions[1,2,3]. Those ncRNAs have a low potential to be translated into proteins and can be sub-divided according to their size into small (200–100,000 nt, lncRNAs). The investigation of patient derived non-small cell lung carcinoma samples revealed decreased PANDAR levels compared to adjacent healthy tissue This correlates with increased tumor size and tumor-nodus-metastasis (TNM) stage[15]. Subsequent functional characterization of interacting candidates revealed a potential role of PANDAR in pre-mRNA splicing regulation via one of its newly identified binding partners polypyrimidine tract-binding protein 1 (PTBP1)
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