A remodeled Pol II complex on RNA template
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Triticum Aestivum (wheat)
TISSUE(S): Plant Cell, Germ
SUBMITTER: 
Tibor Pechan
LAB HEAD: Tibor Pechan
PROVIDER: PXD033736 | Pride | 2022-09-01
REPOSITORIES: Pride
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Project description:Diverse elements within the 5’ untranslated region of an mRNA can influence the translation efficiency at the main AUG codon. We previously identified a core picornaviral like Y16X11-AUG motif with 16-nt polypyrimidine CU-tract separated by an 11-nt spacer sequence from the 13th AUG codon recognized as the preferred initiation site within the Triticum mosaic virus (TriMV) internal ribosome entry site (IRES) element. The motif is proposed to function as an internal ribosomal landing site at the designated start codon. Here we exposed the cooperative role of multiple CU-rich segments flanking the TriMV YX-AUG motif to drive internal initiation of translation at the preferred start site. We propose that these auxiliary domains may enhance the ribosome capacity at proximity of the correct initiation site. These polypyrimidine tracts can be modulated with a cryptic AUG and in a position-dependent manner to replace the native YX-AUG motif and to reprogram translation to the upstream sites, and thus uncovering a new layer of control of the selection of the initiation site. In line with these observations, mass spec analysis of proteins directly interacting with translationally impaired TriMV IRES mutants that bear these motifs indicated an enrichment in 40S and 60S ribosomal related proteins, revealing a new function of polypyrimidine tracts to regulate IRES-driven translation. Accessibility of these RNA regions for in trans interaction was validated by SHAPE analysis of the entire TriMV leader sequence and supported by the ability of anti-sense oligonucleotides designed to block the CU-tracts accessibility to impair IRES activity. This is the first evidence that defines the core modular domains required for start codon selection in a complex, multi-AUG viral 5’UTR for translation in plants.
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Project description:In eukaryotic cells, ribosomal RNA is transcribed by RNA polymerase I (Pol I). To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC). In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3 and Core Factor (CF) composed of subunits Rrn6, Rrn7 and Rrn11. We have determined the cryo-EM structure of a 18-subunit yeast Pol I complex together with CF, Rrn3 and transcription scaffold. To aid in modeling the structure based on the cryo-EM map, we performed crosslinking mass spectrometry.
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Project description:Control of RNA transcription is critical for the development and homeostasis of all organisms, and can occur at multiple steps of the transcription cycle, including RNA polymerase II (Pol II) recruitment, initiation, promoter-proximal pausing, and elongation. That Pol II accumulates on many promoters in metazoans implies that steps other than Pol II recruitment are rate-limiting and regulated 1-6. By integrating genome-wide Pol II chromatin immunoprecipition (ChIP) and Global Run-On (GRO) genomic data sets from Drosophila cells, we examined critical features of Pol II near promoters. The accumulation of promoter-proximal polymerase is widespread, occurring on 70% of active genes; and unlike elongating Pol II within the body of genes, promoter Pol II are held paused by factors like NELF, unable to transcribe unless nuclei are treated with strong detergent. Notably, we find that the vast majority of promoter-proximal Pol II detected by ChIP are paused, thereby identifying the biochemical nature of this rate-limiting step in transcription. Finally, we demonstrate that Drosophila promoters do not have the upstream divergent Pol II that is seen so broadly and prominently on mammalian promoters. We postulate this is a consequence of Drosophila’s extensive use of directional core promoter sequence elements, which contrasts with mammals’ lack of directional elements and prevalence of CpG island core promoters. In support of this idea, we show that the fraction of mammalian promoters containing a TATA box core element is dramatically depleted of upstream divergent transcription. ChIP-seq data set for Pol II (rpb3) (2 replicates).
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