BackgroundAnaerobic, mesophilic, and cellulolyticRuminiclostridium cellulolyticumproduces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by thecip-celoperon inR. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from thecip-celmRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits.ResultsIn this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures incip-celoperon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3′ end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing ofcip-celtranscripts, which can be potentially used to control the stoichiometry of gene expression in an operon.ConclusionsOur findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability incip-celoperon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02357-5.