Poly (A)-rich ribonucleoprotein complexes from HeLa cell messenger RNA

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Polyribosomal messenger RNA (mRNA) in HeLa cells possesses 3'-OH-terminal polyadenylate sequences.
• These poly(A)-rich sequences are associated with tightly bound proteins at the ribonucleoprotein level.

Purpose of the Study:

• To investigate the proteins associated with polyadenylate sequences in HeLa cell polyribosomes.
• To characterize the molecular weight and binding properties of these poly(A)-associated proteins.

Main Methods:

• Affinity chromatography using poly(U)-Sepharose to isolate poly(A)-rich RNA and associated proteins.
• RNase A and T1 digestion of polyribosomes.
• Polyacrylamide gel electrophoresis (PAGE) for protein molecular weight determination.

Main Results:

• Proteins remain associated with poly(A)-rich RNA during affinity chromatography and co-elute with RNA.
• Co-purification requires the molecular integrity of the poly(A) sequence.
• PAGE resolved two protein components of 74,000 and 62,000 molecular weight associated with poly(A).
• The 62,000 MW protein is specific to messenger RNA poly(A), while the 74,000 MW protein is also found in nuclear RNA.

Conclusions:

• Both nuclear and polyribosomal poly(A) sequences in HeLa cells are associated with proteins.
• A 62,000 molecular weight protein is specifically associated with messenger RNA poly(A) sequences.