In:
Science, American Association for the Advancement of Science (AAAS), Vol. 381, No. 6653 ( 2023-07-07)
Abstract:
Discovered in the mitochondrion of Trypanosoma brucei , a protozoan parasite that causes African sleeping sickness, RNA editing denotes a spectrum of phylogenetically widespread and often mechanistically unrelated molecular processes that change RNA sequence. In trypanosomal RNA editing, gRNAs direct massive recoding of cryptic mitochondrial transcripts to generate mRNAs. Assembled into the partially defined editosome, two principal ribonucleoprotein particles execute an unconventional method of genetic information transfer from gRNA to mRNA. The RNA-editing substrate-binding complex (RESC) stabilizes gRNAs and engages mRNAs. The RNA-editing catalytic complex (RECC) fulfills gRNA-programmed mRNA cleavage, uridine insertion or deletion, and religation reactions. RATIONALE Mitochondrial gRNA forms an imperfect duplex with mRNA precursor in which secondary structure defines multiple editing sites. To reveal gRNA stabilization and mRNA recognition mechanisms, we have determined atomic structures of three states of the RESC using cryogenic electron microscopy and characterized individual subunits’ RNA-binding specificity. RESULTS Biochemical studies defined the RESC as a heterogenous assembly of ~18 proteins that enclose gRNAs and mRNAs. Prior work also demonstrated that the RESC1/2 dimer stabilizes gRNAs and most other proteins bind mRNAs. The structure of the six-member RESC-A shows how RESC2 RNA triphosphatase pseudoenzyme engulfs the triphosphorylated gRNA’s 5′ end, and how the RESC5/6 dimer fastens the 3′ end. These contacts promote gRNA folding into a “hairpin-like” conformation and shield both termini from nucleases. The 10-polypeptide RESC-B structure suggests that a remodeling event recovers gRNA from the RESC-A “storage” mode and transitions the single-stranded molecule into mRNA proximity. In the process, the gRNA’s 5′ end is ejected from the RESC2 triphosphate binding tunnel but the 3′ end remains wedged between RESC5 and RESC6, which are the only proteins shared between RESC-A and RESC-B. All RESC-B subunits, including RESC5/6, contact mRNA along a ~20-nucleotide segment. However, gRNA and mRNA do not interact within RESC-B boundaries. A typical gRNA starts with an “anchor” fully complementary to the mRNA target; the adjacent “guiding” part pairs with mRNA sparsely and creates editing sites typified by single-stranded bulges and loops. A few nucleotides separate this “information-rich” sequence from the terminal uridine tail. Mechanistically, sequestering gRNA’s “information-poor” 3′ end inside RESC-B allows guiding and anchor parts to hybridize with mRNA beyond RESC-B’s surface. Apparently, RESC-B proteins recruit gRNAs and mRNAs irrespective of their sequences and position the two strands in a roughly antiparallel orientation, but distant enough to prevent spurious annealing within the complex. The exposed gRNA and mRNA regions likely sample each other until productive hybridization creates a substrate for the catalytic RECC complex. CONCLUSION The architectures of distinct RESC states reveal a diversity of protein folds that have been co-opted into the RNA editing machinery. We have discovered how common gRNA elements function in stabilizing this short molecule and engaging mRNA. Our results show that gRNA-mRNA recognition emanates from ribonucleoprotein complex remodeling rather than initiating base pairing of the anchor sequence with the mRNA target. Last, structural information on essential functional features, such as the RESC2 triphosphate binding site, may facilitate development of new trypanocides. Structures of RESCs. (Left) RESC-A sequesters gRNA termini, promoting hairpin formation and blocking mRNA access. (Right) RESC-A conversion into RESC-B unfolds gRNA and allows mRNA recognition, likely exposing editing sites to RECC-embedded enzymes.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.adg4725
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2023
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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