3-Untranslated region stem-loop structures are major determinants of chloroplast mRNA stability. The 3 stem-loop region of spinach petD precursor mRNA (pre-mRNA), a chloroplast gene encoding subunit IV of the cytochrome b 6 ⅐f complex, forms a stable RNA-protein complex in vitro with chloroplast stem-loop binding proteins (CSPs) of 55, 41, and 29 kDa. We have previously purified CSP41 and cloned the corresponding cDNA. In vitro studies demonstrated that CSP41 is a bifunctional protein that displays both endoribonuclease and RNAbinding activities. In this work, the RNase activity of CSP41 is further characterized using the bacterially expressed protein. Our data show that CSP41 cleaves both single-stranded and double-stranded RNAs but not DNA. However, it exhibits a preference for stem-loopcontaining RNAs. When the 3-untranslated region of petD pre-mRNA is provided as a substrate, CSP41 specifically cleaves it within the stem-loop region, implying that CSP41 has an important role in the control of petD mRNA stability. Our data also show that the sequencespecific RNA-binding activity of CSP41 affects the rate, but not the specificity, of its RNase activity, suggesting that CSP41 is probably involved in other events of chloroplast RNA metabolism in addition to RNA degradation. By analyzing C-terminal deletions of CSP41, the RNase domain was located between amino acid residues 73 and 191.Most chloroplast precursor mRNAs (pre-mRNAs) and mature mRNAs contain an inverted repeat sequence in their 3Ј-untranslated region (UTR) 1 that can fold into a stable stemloop structure. Both in vitro and in vivo studies have demonstrated that these 3Ј stem-loop structures are required for correct 3Ј processing of pre-mRNAs and for stabilization of mature mRNAs, by impeding processive 3Ј to 5Ј exonucleolytic degradation. In the absence of a stable secondary structure, or by readthrough of the stem-loop structure, mRNA becomes heterogeneous and generally unstable (1-7). Therefore, the 3Ј stem-loop structure has a primary role in controlling chloroplast mRNA accumulation through pre-mRNA processing and the protection of upstream sequences. Removal of the 3Ј stemloop structure, therefore, is likely to be a key regulatory step in chloroplast mRNA degradation.Bacterial pre-and mature mRNAs are another major group of mRNAs containing 3Ј-inverted repeats. The 3Ј stem-loop structures of bacterial mRNAs function in the processing of pre-mRNAs and the stabilization of mature mRNAs by a mechanism similar to that found in chloroplasts (8). Extensive studies have shown that at least three groups of proteins are involved in the control of 3Ј to 5Ј degradation of bacterial mRNAs: 1) endoribonucleases, such as RNase E and RNase III, which cleave the 3Ј-UTR within or upstream of the stem-loop structure to overcome the secondary structure barrier; 2) exoribonucleases, such as RNase II and polynucleotide phosphorylase, which bind to the free 3Ј end created by the endoribonucleases and degrade mRNA in the 3Ј to 5Ј direction; and 3) stem-loopbinding pr...