Visualization of a group II intron in the 23S rRNA of a stable ribosome.
Thousands of introns have been localized to rRNA genes throughout the three domains of life. The consequences of the presence of either a spliced or an unspliced intron in a rRNA for ribosome assembly and packaging are largely unknown. To help address these questions, and to begin an intron imaging study, we selected a member of the self-splicing group II intron family, which is hypothesized to be the progenitor not only of spliceosomal introns but also of non-LTR retrotransposons. We cloned the self-splicing group II Ll.LtrB intron from Lactococcus lactis into L. lactis 23S rRNA. The 2,492-nt Ll.LtrB intron comprises a catalytic core and an ORF, which encodes a protein, LtrA. LtrA forms a ribonucleoprotein (RNP) complex with the intron RNA to mediate splicing and mobility. The chimeric 23S-intron RNA was shown to be splicing proficient in its native host in the presence of LtrA. Furthermore, a low-resolution cryo-EM reconstruction of the L. lactis ribosome fused to the intron-LtrA RNP of a splicing-defective Ll.LtrB intron was obtained. The image revealed the intron as a large, well defined structure. The activity and structural integrity of the intron indicate not only that it can coexist with the ribosome but also that its presence permits the assembly of a stable ribosome. Additionally, we view our results as a proof of principle that ribosome chimeras may be generally useful for studying a wide variety of structured RNAs and RNP complexes that are not amenable to NMR, crystallographic, or single-particle cryo-EM methodologies.
Slagter-Jäger
Jacoba G
JG
Center for Medical Science, Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY 12208, USA.
Allen
Gregory S
GS
Smith
Dorie
D
Hahn
Ingrid A
IA
Frank
Joachim
J
Belfort
Marlene
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D020285:Cryoelectron Microscopy; D007438:Introns; D013294:Lactococcus lactis / Q000235:genetics; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000648:ultrastructure*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000648:ultrastructure*; D017381:Spliceosomes / Q000737:chemistry / Q000648:ultrastructure*
D001426:Bacterial Proteins; D012338:RNA, Ribosomal, 23S; D012261:Ribonucleoproteins; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase