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

Abstract

DMS (dimethylsulfate) is a time-tested chemical probe for nucleic acid secondary structure that has recently re-emerged as a powerful tool to study RNA structure and structural changes, by coupling it to high throughput sequencing techniques. This variant, termed DMS-MaPseq, allows for mapping of all RNAs in a cell at the same time. However, if an RNA adopts different structures, for example during the assembly of an RNA-protein complex, or as part of its functional cycle, then DMS-MaPseq cannot differentiate between these structures, and an ensemble average will be produced. This is especially challenging for long-lived RNAs, such as ribosomes, whose steady-state abundance far exceeds that of any assembly intermediates, rendering those inaccessible to DMS-MaPseq on total RNAs. These challenges can be overcome by purification of assembly intermediates stalled at specific assembly steps (or steps in the functional cycle), via a combination of affinity tags and mutants stalled at defined steps, and subsequent DMS probing of these intermediates. Interpretation of the differences in DMS accessibility is facilitated by additional structural information, e.g. from cryo-EM experiments, available for many functional RNAs. While this approach is generally useful for studying RNA folding or conformational changes within RNA-protein complexes, it can be particularly valuable for studying the role(s) of DEAD-box proteins, as these tend to lead to larger conformational rearrangements, often resulting from the release of an RNA-binding protein from a bound RNA. Here we provide an adaptation of the DMS-MaPseq protocol to study RNA conformational transitions during ribosome assembly, which addresses the challenges arising from the presence of many assembly intermediates, all at concentrations far below that of mature ribosomes. While this protocol was developed for the yeast S. cerevisiae, we anticipate that it should be readily transferable to other model organisms for which affinity purification has been established.

Keywords: Affinity purification; DEAD-box proteins; DMS; MapSeq; Ribosome assembly.

Figure 1:. Selecting affinity tags and stalling mutations to isolate homogeneous samples.

(A) Cartoon illustrating different 40S assembly intermediates and their binding to UtpA (which contains Utp10) and Dim1 assembly factors. Both UtpA and Dim1 will purify a series of intermediates, only a subset of which are shown here. (B) A split tag approach increases the homogeneity of the sample, as illustrated here with samples that contain both Dim1 and Dhr1. Note that this panel focuses in on molecules containing Dim1 (and thus has additional intermediates not shown in A for clarity) to illustrate how a split-tag can reduce the heterogeneity of the sample. The enriched intermediate is highlighted with a circle. (C) Combining an affinity tag on a factor like Rio2 which co-purifies a series of intermediates (characterized by different bound assembly factors, such as Ltv1, Enp1 and Rio2), but enriches an early cytoplasmic intermediate (in the red circle), with a mutation that leads to stalling (like the Rps15_YRR mutation), allows for the isolation of an intermediate that would otherwise be transient (in the blue circle) at homogeneity sufficient for RNA structure probing.

Figure 2:

Schematic of the DMS MaPseq approach with purified assembly intermediates.

Figure 3:. DMS MaPseq of 40S ribosomes produces few highly exposed nucleotides.

(A) Distribution of DMS accessibility values of nucleotides in mature 40S subunits from [10] into the bins as assigned by [44]. Compare to Figure 2D&F in [44]. (B) Mapping of DMS accessibility values [10] onto the 3D structure of mature 40S ribosomes (pdb ID: 3jam). Binning is done according to our criteria: red (highly exposed residues: >4); orange (2-4); yellow (1-2); blue (0.5-1); fully protected residues (<0.5) are not shown.