During starvation induced encystment, cells of the myxomycete Didymium iridis accumulate a 7.5-kb RNA that is the result of alternative processing of pre-rRNA. The 5¢ end corresponds to an internal processing site cleaved by the group I-like ribozyme DiGIR1, located within the twinribozyme intron Dir.S956-1. The RNA retains the majority of Dir.S956-1 including the homing endonuclease gene and a small spliceosomal intron, the internal transcribed spacers ITS1 and ITS2, and the large subunit rRNA lacking its two group I introns. The formation of this RNA implies cleavage by DiGIR1 in a new RNA context, and presents a new example of the cost to the host of intron load. This is because the formation of the 7.5-kb RNA is incompatible with the formation of functional ribosomal RNA from the same transcript. In the formation of the 7.5-kb RNA, DiGIR1 catalysed cleavage takes place without prior splicing performed by DiGIR2. This contrasts with the processing order leading to mature rRNA and I-DirI mRNA in growing cells, suggesting an interplay between the two ribozymes of a twinribozyme intron.Keywords: Didymium iridis; group I intron; ribozyme; prerRNA processing.Group I introns contain a conserved set of sequences and structural elements that are involved in the removal of the intron by splicing. They constitute one class out of fewer than 10 classes of naturally occurring ribozymes [1]. Group I introns vary considerably in complexity. Most introns contain only the sequence information required for splicing, whereas others contain large extensions of the peripheral domains. Some of the larger group I introns contain an open reading frame, usually represented by a homing endonuclease gene (HEG). HEGs are found in different configurations, e.g. fused in frame with the upstream exon or as an independent expression unit [2]. The most complex group I introns are the twin-ribozyme introns that in addition contain a group I-like cleavage ribozyme (GIR1) involved in the expression of the intron HEG [3].The complex structure of the twin-ribozyme introns suggests a complex biology. This has been demonstrated in the case of the Dir.S956-1 (former DiSSU1; the recently introduced nomenclature for group I introns [4] is used throughout this paper) intron found in the small subunit ribosomal RNA (SSU rRNA) gene in the myxomycete Didymium iridis (Fig. 1). One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation (Fig. 1, left panel). In addition, this ribozyme displays a pronounced 3¢ splice site hydrolysis activity, which induces the formation of full-length intron RNA circles using a processing pathway that is distinctly different from splicing ([5]; unpublished data]. The other ribozyme (DiGIR1), which along with the I-DirI HEG is inserted in DiGIR2, carries out hydrolysis at two internal processing sites (IPS1 and IPS2) located at its 3¢ end [5,6]. In vivo, this cleavage results in the formation of the 5¢ end of the I-DirI mRNA and is followed by cleavage at an in vivo specific internal processing site (IPS3) dow...