ABSTRACT
Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP- ribosylation enzyme and plays important roles in a variety of cellular processes, including DNA damage response and tumor development. However, the post-transcriptional regulation of PARP1 remains largely unknown. In this study, we identified that the mRNA of PARP1 is associated with nuclear factor 90 (NF90) by RNA immunoprecipitation plus sequencing (RIP-seq) assay. The mRNA and protein levels of PARP1 are dramatically decreased in NF90-depleted cells, and NF90 stabilizes PARP1's mRNA through its 3'UTR. Moreover, the expression levels of PARP1 and NF90 are positively correlated in hepatocellular carcinoma (HCC). Finally, we demonstrated that NF90-depleted cells are sensitive to PARP inhibitor Olaparib (AZD2281) and DNA damage agents. Taken together, these results suggest that NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma cells, and NF90 is a potential target to inhibit PARP1 activity.
Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Nuclear Factor 90 Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , RNA Stability , RNA, Messenger/metabolism , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Humans , Liver Neoplasms/metabolism , Nuclear Factor 90 Proteins/isolation & purification , RNA, Messenger/geneticsABSTRACT
Long noncoding RNAs (lncRNAs) are nonprotein coding transcripts longer than 200 nucleotides. Recently in mammals, thousands of long noncoding RNAs have been identified and studied as key molecular players in different biological processes with protein complexes. As a long noncoding RNA, maternally expressed gene 3 (MEG3) plays an important role in many cellular processes. However, the mechanism underlying MEG3 regulatory effects remains enigmatic. By using the specific interaction between MS2 coat protein and MS2 RNA hairpin, we developed a method (MS2-tagged RNA affinity purification and mass spectrometry (MTRAP-MS)) to identify proteins that interact with MEG3. Mass spectrometry and gene ontology (GO) analysis showed that MEG3 binding proteins possess nucleotide binding properties and take part in transport, translation, and other biological processes. In addition, interleukin enhancer binding factor 3 (ILF3) and poly(A) binding protein, cytoplasmic 3 (PABPC3) were validated for their interaction with MEG3. These findings indicate that the newly developed method can effectively enrich lncRNA binding proteins and provides a strong basis for studying MEG3 functions.