Using DMS-MaPseq to uncover the roles of DEAD-box proteins in ribosome assembly

Liu, Xin; Huang, Haina; Karbstein, Katrin

doi:10.1016/j.ymeth.2022.05.001

Cited by 5 publications

(5 citation statements)

References 56 publications

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“…DMS MaPseq RNA Library Preparation. RNA library preparation was adapted from [44] and performed as described [22,38]. Briefly, RNAs were fragmented with the addition of 20 mM MgCl 2 at 94 °C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…To better understand the molecular basis for the loss of many head RPs and determine whether additional rRNA regions are misfolded in the ribosomes from the L216S mutant yeast, we utilized DMS-MaPseq to probe the structure of mature 40S subunits from yeast expressing wild type Ltv1 or L216S Ltv1. As previously described [22,38], ribosomes were exposed to DMS or mock treatment, and after quenching the reaction, 18S rRNA was fragmented, reverse transcribed, and sequencing libraries prepared. After sequence alignment, modified nucleotides are misread in the reverse transcription reaction and appear as a "mutation" relative to the genomic DNA (Figure S3A).…”

Section: Dms-mapseq Reveals Global Roles For Ltv1 In 40s Structurementioning

confidence: 99%

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A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

Blomqvist,

Huang,

Karbstein

2023

Preprint

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Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. Our data demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly.

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Section: Methodsmentioning

confidence: 99%

Section: Dms-mapseq Reveals Global Roles For Ltv1 In 40s Structurementioning

confidence: 99%

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

Blomqvist,

Huang,

Karbstein

2023

Preprint

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show abstract

“…RNA library preparation was adapted from [ 48 ] and performed as described [ 22 , 41 ]. Briefly, RNAs were fragmented with the addition of 20 mM MgCl 2 at 94°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…To better understand the molecular basis for the loss of many head RPs and determine whether additional rRNA regions are misfolded in the ribosomes from the L216S mutant yeast, we utilized DMS-MaPseq to probe the structure of mature 40S subunits from yeast expressing wild type Ltv1 or L216S Ltv1. As previously described [22,41], ribosomes were exposed to DMS or mock treatment, and after quenching the reaction, 18S rRNA was fragmented, reverse transcribed, and sequencing libraries prepared. After sequence alignment, modified nucleotides are misread in the reverse transcription reaction and appear as a "mutation" relative to the genomic DNA (S4A Fig) . Thus, the mutational rate at each nucleotide is a measure of its propensity to be modified by DMS.…”

Section: Dms-mapseq Reveals Global Roles For Ltv1 In 40s Structurementioning

confidence: 99%

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

Blomqvist,

Huang,

Karbstein

2023

PLoS Genet

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Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. A combination of data from yeast genetics, mass spectrometry, DMS probing and biochemical experiments demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly.

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“…This linker was used to prime reverse transcription, creating cDNAs that were subsequently converted into sequencing libraries using 18S rRNA-specific primers for analysis of 18S rRNA 3 0 -ends. Due to concerns that reverse transcription through the Dim1-dimethylation site in 18S rRNA (m 6 2 A1781 and m 6 2 A1782) would pose complications [67,68], we used a yeast strain containing a mutation in Dim1 (Dim1-E85A) that does not dimethylate 18S rRNA [69], thus allowing us to sequence the final 40 to 60 nucleotides of 18S rRNA. Control experiments indicate that this does not affect the frequency of miscleavage (S2C Fig) . We obtained 2.8 to 4 million reads per sample, with 96% to 99% of reads aligning to the 3 0 -end of 18S rRNA.…”

Section: Nob1 Miscleaves Pre-18s Rrnamentioning

confidence: 99%

The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage

Parker,

Brunk,

Getzler

et al. 2024

PLoS Biol

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The 18S rRNA sequence is highly conserved, particularly at its 3′-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3′-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3′-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3′-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

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Using DMS-MaPseq to uncover the roles of DEAD-box proteins in ribosome assembly

Cited by 5 publications

References 56 publications

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head

The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage

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