Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme

Abstract

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 59 end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent selfcleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

In vivo Expression of a Group I Intron HEG from the Antisense Strand of Didymium Ribosomal DNA

“…While a product of the expected length for I50 spliced I-DirII RNA was obtained with OP773, the use of OP98 resulted in a longer DNA-type PCR product that contained I50 (data not shown), indicating that the 5' end of the transcript is located within 20 nt immediately upstream of the I-DirII AUG start codon. Similar short 5' untranslated regions are common among the His-Cys homing endonuclease mRNAs, and range in length from 9-13 nt for all the reported messengers 8,10,18 except 58 nt for I-DirI mRNA. 3,5 To test whether the sequence immediately upstream the I-DirII HEG could function as a promoter for RNA pol II, a 248 bp , which is identical to the specific sequence at the 5' end of �P41 (see Table 1), was used as downstream primer to PCR amplify the cDNA reverse transcribed from �P41.…”

“…6,7 Here, the I-NaeI HEG expression is also dependent on a group I-like lariat-forming ribozyme (NaGIR1) within the intron (our unpublished results). 8 Finally, the I-PpoI HEG, found within the Ppo.L1925 group I intron in Physarum polycephalum, is transcribed by RNA pol I and translated from an mRNA corresponding to the excised linear group I intron RNA. [9][10][11] Dir.S956-2 is a complex HEG-containing group IC1 intron located within the small subunit (SSU) rDNA of the D. iridis Costa Rica 8 isolate 12 at the exact same location as Dir.S956-1.…”

A natural fast-cleaving branching ribozyme from the amoeboflagellateNaegleria pringsheimi

“…2B and Table S1). In vitro, GIR1 catalyzes two types of cleavage reactions, the natural branching reaction 13,21 and hydrolytic cleavage at IPS which is considered to be an in vitro artefact. 5 The two reactions can be distinguished by primer extension analysis of the 3' cleavage product because the lariat cap resulting from the branching reaction leads to a primer extension stop signal three nucleotides downstream of the IPS.…”

A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA

“…2A, ''2A''). Cleavage by transesterification (branching) is the resulting reaction in vivo (Vader et al 1999;Decatur et al 2000), and cleavage by hydrolysis is considered an in vitro artifact (Nielsen et al 2005). To expand on the importance of the ligation reaction in cleavage analyses, a ligation experiment with 39-fragments of varying length was carried out.…”