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Validation of protein-RNA interaction using the protein microarray

ABSTRACT: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas. Tests on the HuProt protein microarray have showed that proteins are folded in their native conformation and retain function, such as protein-RNA binding, protein-DNA binding, etc. Therefore, the HuProt protein microarray can be an alternative tool to study the RNA-binding activities of proteins. We generated a pool of Cy5-labelled RNAs by mixing the fragmented total RNAs (13.5%) and in vitro transcribed RNAs (86.5%). To amplify the non-ribosomal RNAs (non-rRNAs), we generated the cDNA by reverse transcription using the total RNAs of HEK293 cells from which the ribosomal RNAs were first removed as the template; we then amplified the double-stranded templates of the T7 in vitro transcription by PCR using random primers; the RNAs were finally produced by the T7 in vitro transcription. We randomly labeled the Cy5 dye to the fragmented total RNAs of HEK293 cells and the in vitro transcribed RNAs above. The calculated Cy5 labeling efficiency was 1 dye per 110nt for the fragmented total RNAs and 1 dye per 170nt for the in vitro transcribed RNAs. The pooled RNAs covered different types of RNAs with a high complexity, particularly non-rRNAs. To investigate the RNA-protein interactions, we incubated the Cy5-labelled RNAs with protein microarrays and quantified the fluorescence signals on the microarrays after several washing steps.

ORGANISM(S): Homo sapiens (human)

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PROVIDER: S-BSST1172 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: A novel human nuclear receptor interaction protein (NRIP) has recently been discovered by Chen SL et al, which may play a role in enhancing the transcriptional activity of steroid nuclear receptors in prostate (LNCaP) and cervical (C33A) cancer cell lines. However, knowledge about the biological functions and clinical implications of NRIP, is still incomplete. Our aim was to determine the distribution of NRIP expression and to delineate the cell types that express NRIP in various malignant tumors and healthy non-pathological tissues. This information will significantly affect the exploration of its physiological roles in healthy and tumor cells. METHODS: By using tissue microarray (TMA) technology and an anti-NRIP monoclonal antibody immunohistochemical (IHC) survey, NRIP expression was examined in 48 types of tumors and in a control group of 48 matched or unmatched healthy non-neoplastic tissues. RESULTS: Our survey results showed that ten cases were revealed to express the NRIP in six malignancies (esophageal, colon, breast, ovarian, skin, and pancreatic cancers), but not all of these specific tumor types consistently showed positive NRIP expression. Moreover, malignant tumors of the stomach, prostate, liver, lung, kidney, uterine cervix, urinary bladder, lymph node, testis, and tongue revealed no NRIP expression. Among the control group of 48 matched and unmatched non-neoplastic tissues, all of them demonstrated IHC scores less than the cut-off threshold of 3. In addition, ten cores out of thirty-six carcinomatous tissues revealed positive NRIP expression, which indicated that NRIP expression increases significantly in carcinoma tissue cores, comparing to the matched controlled healthy tissues. CONCLUSION: This is the first study to use a human TMA and IHC to validate the nuclear localization for this newly identified NRIP expression. In considering the use of NRIP as a potential diagnostic tool for human malignancies survey, it is important to note that NRIP expression carries a sensitivity of only 23%, but has a specificity of 100%. There is also a significant difference in positive NRIP expression between primary carcinomatous tissues and matched controlled healthy tissues. Although further large-scale studies will merit to be conducted to evaluate its role as a potential adjunct for cancer diagnosis, data from this study provides valuable references for the future investigation of the biological functions of NRIP in humans.

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Validation of protein-RNA interaction using the protein microarray

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