Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

  • Yangzi He, Danmark
Ribonucleic acids (RNAs) take centre stage in gene expression. In eukaryotes, most RNAs are transcribed as precursors, and these precursors are co- or post-transcriptionally processed and assemble with particular proteins to form ribonucleoproteins (RNPs). Mature RNPs participate in various gene expression events, are then subject to recycling, disassembly or degradation. RNA helicases are highly conserved enzymes that use ATP to bind or remodel RNA or RNPs. They function in nearly every aspect of eukaryotic RNA metabolism.
The spliceosome catalyzes pre-mRNAs splicing, which removes introns and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH/RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained.
The ribosome translates the genetic message encoded in mRNAs to synthesize proteins. Initiation of translation requires localization and recognition of the start codon at the P-site of the 40S small ribosomal subunit. On most eukaryotic mRNAs, the start codon is identified by a scanning mechanism, whereby a small subunit loaded with an initiator methionyl-tRNA binds to the 5’ cap-proximal region of mRNAs, and goes through a base-by-base inspection of the 5’ untranslated region (5’ UTR) in the 5’ to 3’ direction for an AUG initiation codon. Scanning of 5’ UTRs containing even weak secondary structures requires eukaryotic initiation factor (eIF)4A, a DEAD-box RNA helicase, as well as ancillary factors eIF4B and eIF4G. In higher eukaryotes, scanning of mRNAs containing stable secondary structures in the 5’ UTR furthermore requires DHX29, another DEAH/RHA helicase. I participated in characterizing the synergistic activation of eIF4A by eIF4B and eIF4G and successfully purified DHX29 for structural studies.
OriginalsprogEngelsk
ForlagAarhus University, Faculty of Science and Technology
Antal sider174
StatusUdgivet - 2012

Note vedr. afhandling

In his PhD research, Yangzi He used X-ray crystallography to investigate the structure of Prp43, a yeast RNA helicase of the DEAH/RHA family that functions in pre-mRNA splicing and ribosome biogenesis. This study yielded the first high-resolution structure of this important class of enzymes. Yangzi He also studied the structure and mechanism of eIF4A and DHX29, two human RNA helicases required for the initiation of protein synthesis

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