Three of the four rRNAs are transcribed as 35S pre-RNA by RNA polymerase I (Kressler et al., 1999). These results suggest that the ribosome can form an active RNP complex spontaneously in vitro (Hosokawa et al., 1966). Cryo-electron microscopy has provided a straightforward understanding of the structural differences between eukaryotic and prokaryotic ribosomes (Spahn et al., 2004).
The MUDPIT approach has successfully identified many known ribosome core components (Link et al., 1999). In contrast, mammalian RACK1 was excluded from Asc1p-containing wild-type yeast ribosomes (Gerbasi et al., 2004). The ribosome was positioned directly upstream of the start codon in a reconstituted in vitro translation system (Pestova et al., 1998).
They hypothesized that a number of ITAFs could bind to and stimulate the ODC IRES ( Pyronnet et al., 2005 ). In a previous study, an increase in specific mRNA levels was observed in asc1∆ null strains compared to wild-type strains (Hoffmann et al., 1999). A precedent for such phenomena has recently been reported for the ribosomal protein L13A (Mazumder et al., 2003).
Mass spectra corresponding to ribosomal proteins from the wild-type and asc1∆ null strains were obtained using MUDPIT ( Link et al., 1999 ; Washburn et al., 2001 ).
Temp (Celcius) Temp (Celcius) 30 39 Wild-type
Ssb1/2p has been determined to bind to the ribosome in a conformation-dependent manner ( Pfund et al., 1998 ). Recently, a role for one of the HSP70 chaperones in ribosome biogenesis was shown (Meyer et al., 2007). Mutation of a pyrimidine (PY) tract in the 5′UTR of the ODC mRNA has been shown to inhibit IRES activity ( Pyronnet et al., 2000 ).
The mutant ODC IRES has been shown to suppress translational activity compared to the wild-type IRES ( Pyronnet et al., 2000 ). In the top sample, the wild-type form of the ODC caused a control response. Proteins identified from the wild-type (left side) and mutant (right side) RNA affinity capture reactions were displayed using BIGCAT's Clusterer visualization application ( McAfee et al., 2006 ).
Our data strongly suggest that PCBP2 and ZNF9 function as ITAFs of the ODC IRES. We found that two nucleic acid binding proteins PCBP2 and ZNF9 bind to this region of the ODC IRES. Furthermore, we found that both PCBP2 and ZNF9 increase ODC IRES activity.
Therefore, it is possible that additional ITAFs interact with the ODC IRES element independently of the PY tract by binding to the RNA sequence shared by both wild-type and mutant ODC IRES (Pyronnet et al. , 2000). Our proteomic screen revealed that PCBP2 and ZNF9 bind to the active form of the ODC IRES RE, but not an inactive form. Finally, supplementation of asc1-deficient yeast with mammalian RACK1 also restored protein levels back to the wild-type state.
However, a common feature of CLIPS is their shared biochemical association with the ribosome (Albanese et al., 2006). In addition, another member of the HSP chaperone family controls yeast ribosome biogenesis (Meyer et al., 2007). Previous studies have shown that Ssb1/2p binds to the ribosome in a conformation-dependent manner (Pfund et al., 1998).
However, the 18S rRNA (which is included in the 40S subunit) is partially processed in the cytoplasm (Kressler et al., 1999; Warner, 2001). The drug suppresses the synthesis of proteins encoding ribosome components and ribosome biogenesis factors (Guertin et al., 2006; Tang et al., 2001).
Conclusion: Asc1p is part of the ribosome core and facilitates ribosome biogenesis
Therefore, if Asc1p is critical for docking of eIF3 to the ribosome, then one would expect asc1-deficient yeast to exhibit a severe growth defect at 30 °C. Consistent with ASC1 playing an important role in translation, asc1-deficient yeast does not grow at 39°C. This defect is primarily due to asc1-deficient yeast not forming nascent ribosomes at 39°C.
Because asc1-deficient yeast appears to have a similar loss of all ribosome species at the nonpermissive temperature, it is unlikely that ASC1 functions to stimulate translational initiation. Future studies outlined above will define the precise mechanistic contribution of ASC1 to translational repression and ribosome biogenesis.
Discovery of novel IRES RNP complexes
Mass spectrometry analysis of wild-type and mutant RNA-binding proteins showed that two proteins bound abundantly and specifically to the wild-type IRES RNA. Further biochemical experiments suggested that ZNF9 and PCBP2 were bona fide ODC IRES binding proteins. Further, loss-of-function and overexpression analyzes suggested that PCBP2 and ZNF9 function to stimulate ODC IRES cap-independent translation.
Applying a simplistic model of hereditary disease to the ZNF9 gene, it is possible that mutations in ZNF9 disrupt cap-independent mRNA translation in type 2 myotonic dystrophy. Importantly, mice that are heterozygous mutants of ZNF9 share many of the human symptoms of myotonic dystrophy, including: myotonic discharges, cardiac conduction. Therefore, haploinsufficiency of ZNF9 in mice causes phenotypes consistent with myotonic dystrophy in humans.
The nature of the ZNF9 mutation has led to some disease models that ignore the gene's natural function in disease etiology. Similarly, patients with type-1 myotonic dystrophy have tandem CTG expansions in a noncoding region of the DMPK gene (Brook et al., 1992; Carango et al., 1993). Therefore, the genes causing both forms of myotonic dystrophy harbor similar mutations in genes predicted to have divergent functions.
If this is the case, it would explain why CTG and CCTG expansion mutations occur in genes with different functions but have the same result; development of symptoms similar to myotonic dystrophy. The RNA toxicity model of myotonic dystrophy assumes that CUG-binding proteins are sequestered in the nuclei of patients with the disease. Because there are additional forms of myotonic dystrophy for which the affected genes are unknown, this approach may pinpoint the etiology of these other dystrophy type(s).
A number of the proteins that bind to both wild-type and mutant (inactive) IRES RNA are established ITAFs. Therefore, a number of the proteins identified in our screen bind to the wild-type and inactive ODC IRES and are established ITAFs. I raised the possibility that these proteins could also function as ITAFs of the ODC IRES.
Conclusion: ZNF9 and PCBP2 are ITAFs that stimulate the ODC IRES IRES
Localization of a target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli. Translational regulation of the Escherichia coli ribosomal protein L11 operon: mRNA target site analysis using oligonucleotide-directed mutagenesis. Asc1p, a WD40 domain-containing adapter protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes.
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Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome. Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of the La autoantigen in XIAP translation. Analysis of the protein composition of yeast ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis.
The ribosomal protein Rps15p is required for nuclear egress of 40S subunit precursors in yeast. The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosomal nascent chain complex. Cloning of the intracellular receptor for protein kinase C: a homologue of the beta subunit of G proteins.
Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass. Comparison of the effects of an ornithine decarboxylase inhibitor on the intestinal epithelium and on intestinal tumors. Location of the internal ribosome entry site in the 5' noncoding region of the immunoglobulin heavy chain binding protein (BiP) mRNA: evidence for specific RNA-protein interactions.
