A Dual Inhibitory Mechanism Restricts msl-2 mRNA Translation for Dosage Compensation in Drosophila

Beckmann, Karsten; Grs̆ković, Marica; Gebauer, Fátima; Hentze, Matthias W.

doi:10.1016/j.cell.2005.09.016

Cited by 35 publications

(70 citation statements)

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“…We first tested the specific RNP capture protocol in vitro, using the well-studied interaction between Drosophila Sex lethal (Sxl) and the polyuridine (U) stretches located within the 5 ′ (A and B site) and 3 ′ UTR (E and F sites) of the male specific lethal-2 (msl-2) mRNA (Gebauer et al 2003;Beckmann et al 2005). The interaction of Sxl with poly(U) motifs has been well-characterized biochemically and structurally (Inoue et al 1990;Valcárcel et al 1993;Handa et al 1999;Hennig et al 2014).…”

Section: Specific Rnp Capture In Vitromentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

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Section: Specific Rnp Capture In Vitromentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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“…Several well-characterized TL features can repress translation to varying degrees. Inhibitory features include upstream open reading frames, which divert ribosomes from the main open reading frame of an mRNA (Hinnebusch 2005;Arribere and Gilbert 2013;Pelechano et al 2013); stable RNA secondary structures, which block or impede ribosome recruitment or scanning (Kozak 1990;Ding et al 2014;Weinberg et al 2016); and target sites for certain RNA-binding proteins, which lead to translational repression and mRNA decay of specific sets of messages (Hentze et al 2004;Beckmann et al 2005). In contrast, relatively little is known about TL features that can act as general translational enhancers, although such elements could contribute substantially to the wide range of TEs observed in eukaryotic cells (Gilbert 2010;Shatsky et al 2014;Lee et al 2015).…”

Section: Introductionmentioning

confidence: 99%

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Zinshteyn

Rojas-Durán

Gilbert

2017

RNA

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Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs.

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“…Translational repression requires SXL binding to specific U-rich sequences in both the 59 and 39 UTRs of msl-2 mRNA. SXL binding to the 39 UTR is thought to inhibit the recruitment of the small ribosomal subunit to the mRNA, while SXL binding to the 59 UTR blocks the scanning toward the AUG initiation codon of those subunits that presumably have escaped control through the 39 UTR (Beckmann et al 2005). How SXL inhibits these steps of translation initiation is unknown.…”

Section: Introductionmentioning

confidence: 99%

Functional domains of Drosophila UNR in translational control

Abaza

Gebauer²

2008

RNA

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Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that SXL recruits the protein upstream of N-ras (UNR) to the 39 UTR of msl-2 mRNA. UNR is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). Here, we dissect the domains of UNR required for translational repression and complex formation with SXL and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with SXL and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human UNR can also form a complex with SXL and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that UNR binding to SXL and msl-2 can be distinguished from translation inhibition. Repression by tethered UNR requires residues from both the aminoterminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of UNR resides within the first 397 amino acids of the protein. Our results identify domains and residues required for UNR function in translational control.

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A Dual Inhibitory Mechanism Restricts msl-2 mRNA Translation for Dosage Compensation in Drosophila

Cited by 35 publications

References 0 publications

Specific RNP capture with antisense LNA/DNA mixmers

Specific RNP capture with antisense LNA/DNA mixmers

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

Functional domains of Drosophila UNR in translational control

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