Immunoprecipitation (IP) of protein X from cell lysate using anti-X antibody, followed by RT-PCR for mRNA-Y, indicates the presence of protein X–mRNA-Y complex in vivo. To confirm this interaction, recombinant protein X and in vitro transcribed mRNA-Y are incubated, followed by affinity purification of protein X. However, mRNA-Y was not present in the eluates containing X but rather in the unbound fraction.
Given below are a few statements to explain the observations:
A. Protein X and mRNA-Y are expressed at the same level in the cell, so the interaction is robust.
B. Protein X and mRNA-Y physically interact via a bridge protein/co-factor absent in the purified protein X.
C. mRNA-Y binds to the antibody used in the immunoprecipitation.
D. The mRNA binding domain of the recombinant protein X may have an altered conformation.
Which one of the following is a combination of all correct statements?

Correct option is B

Correct Answer
(b) B and D
Explanation
The interaction between protein X and mRNA-Y observed in vivo but not reproduced in vitro suggests indirect binding or altered binding conditions. The interaction may require an additional bridging protein or co-factor present in cells but absent in the recombinant system, or the recombinant protein X may not adopt the correct conformation needed for RNA binding.
Information booster
• Many RNA–protein interactions in vivo are mediated by multi-protein complexes
• Recombinant proteins may lack post-translational modifications
• Protein folding can differ between cellular and in vitro systems
• Affinity purification tests direct physical interactions
Additional Knowledge
Statement A is incorrect because expression level does not determine physical interaction or its robustness.
Statement C is incorrect because nonspecific binding of mRNA to the antibody would not explain selective co-IP only in vivo and not in vitro.