Abstract
Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.
Highlights
Of the mitoribosome is largely enigmatic and involves numerous assembly factors
Until now there are only four highly-conserved modification sites mapped on the mtLSU: one pseudouridylation at U1397, which is carried out by RPUSD418, and three 2’-O-ribose methylations, which are catalyzed by three methyltransferases, MRM1, MRM2, and MRM315
Based on the conformation and maturation states of the 16S rRNA, our ensemble of intermediate structures purified with the assembly factors MALSU1 and GTPBP10 can be put in a sequential order representing the late maturation path of the human 39S mitoribosome (Fig. 6)
Summary
Of the mitoribosome is largely enigmatic and involves numerous assembly factors. A valine tRNA forms the central protuberance, thereby replacing the 5S rRNA4 Taken together, this suggests a deviating assembly process for the mitochondrial ribosomes. Similar to the more extensively studied bacterial ribosome, the mitochondrial 39S large subunit requires dozens of assembly factors (AFs), such as methyltransferases[15,16,17], pseudouridine synthases[18,19], GTPases[20,21,22,23,24,25], RNA helicases[26,27], and other factors[28,29,30,31]. NSUN4 has been identified as an rRNA m5C methyltransferase for the methylation of 12S rRNA of the mtSSU on position C911 It belongs to the same protein family as the bacterial assembly factor. The NSUN4–mTERF4 complex has been involved in ribosomal subunit joining in mitochondria[16,29,33,34]
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