Purification and characterization of an endoribonuclease from nucleoplasm and nucleoli of HeLa cells.

C N Kwan

doi:10.1016/s0021-9258(17)32952-6

Abstract

An endoribonuclease has been isolated from HeLa cell nuclei. Approximately 70% of the enzyme appears to be nucleolar bound; 30% is in the nucleoplasm. Studies of the purified enzyme reveal that the enzyme is an endonuclease of estimated molecular weight 16,000. It produces oligonucleotides bearing 5'-phosphate end groups. The enzyme degrades poly(C) and poly(U), as well as rRNA and heterogeneous nuclear RNA, Poly(A), double-stranded RNA, and DNA are not cleaved. The enzyme is heat-labile and is inhibited by 10mM Mg2+ and 50 mM NaCl. The enzyme is probably distinct from previously described nuclear endonucleases.

Highlights

The ribosomal RNA, messenger RNA, and transfer RNA of eukaryotic cells are transcribed as larger precursor molecules which are cleaved by ribonucleases into smaller mature products (l-4)
50 ml (50 to 100 mg of protein) of nucleoplasmic or 10 ml (10 to 20 mg of protein) of nucleolar extract were placed on a DEAE-cellulose column
Multiple peaks of enzymatic activities were found by DEAEcellulose fractionation of either the nucleoplasmic or nucleolar extracts (Fig. 1)

Summary

Introduction

The ribosomal RNA, messenger RNA, and transfer RNA of eukaryotic cells are transcribed as larger precursor molecules which are cleaved by ribonucleases into smaller mature products (l-4). A number of ribonucleases have been found in the nuclei of animal cells. Winicov and Perry [5] have reviewed recent work on the enzymological aspects of rRNA’ processing while I have summarized in Table I the known ribnnucleases from nuclei of animal cells. In earlier work concerning enzymatic processing of rRNA, the fractionation of nucleoplasmic and nucleolar ribonuclease activities on DEAE-cellulose columns was described [13]. These fractionations showed the presence of at least three degradative activities. I have continued these studies and report here the purification of one of the ribonucleases

Methods

Results

Conclusion