#ribonucleoprotein

[unbfacts]

[biochemistries]

O-ribose origami

NMR and neutron scattering work at EMBL in Heidelberg, has determined the structure of the RNA-tagging machinery (the box C/D ribonucleoprotein enzyme). The work reveals that folding ribosomal RNA (rRNA) requires a paired tagging sequence, regulating rRNA methylation.

Post-transcriptional modifications (PTMs) are essential to the cell's life cycle, affecting both pre-ribosomal RNA processing and ribosome assembly. This archaeal enzyme uses numerous guide RNAs (shown above in red) as templates for recognition of the rRNA target sites.

During the biosynthesis and processing of the pre-rRNA transcripts post-transcriptional modifications of ribonucleotides occur in functional regions, including intersubunit interfaces, decoding and peptidyltransferase centres. Among the possible modifications, 2'-Oribose methylation was shown to protect RNA from ribonucleolytic cleavage, stabilize single base pairs, serve as chaperone and affect folding at high temperatures. rRNA methylation is essential for both pre-rRNA processing and ribosome assembly, with complete suppression of methylation leading to cell death.

The structure shows that only one pair of proteins (blue) can tag the RNA at a time (i.e. methylate the 2’-O-ribose).

As shown in the schematic above, the substrate sequences inside the complex are reached by methyltransferase for methylation, whereas the external ones are far from all fibrillarin copies and hence cannot be. Fib = fibrillarin, the methyltransferase that is the first actor in pre-(r)RNA processing. Apo complex means the enzyme without RNA (from the Greek ἀπό, away from, as opposed to the holoenzyme, meaning 'whole')

In the apo-complex the guide D9(D) sequences are close to (far from) fibrillarin. Top left, substrate D9 binds first on the same plane as two fibrillarin copies and is methylated; subsequent addition of substrate D causes a conformational switch that brings the guide–substrate D duplexes and two fibrillarin copies on one plane (centre). Right, substrate D is methylated (Fig. 4a, right). Bottom left, substrate D, added first, binds to the guide D sequences far from fibrillarin, where it cannot be methylated; substrate D9, added next, binds to the guide D9 sequences next to two fibrillarin copies and is methylated. Right, the conformational switch takes place and substrate D is methylated as well.

The methylation site on the actual structure of the single stranded RNA used in the sRNP is designated by a star, below: The red and salmon coloured sequences are identical.

Box C/D motifs are used to classify one kind of small nucleolar RNA-protein complexes (snoRNPs), and further introduction to this field can be read at the site of the UCSF McManus lab here.

This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.

Summarising their model rather neatly, the authors propose that in the absence of RNA substrate,

the asymmetric sRNP preferentially assumes a conformation where the fibrillarin associated with the box C'/D' is on the same plane as the RNA. In this conformation, substrate D' can be loaded on the complex, methylated and released. Addition of the other substrate to the complex half-loaded with product RNA triggers a conformational switch, whereby the newly formed duplexes and the corresponding fibrillarin copies move to one plane, allowing the methylation of substrate D. On the other hand, binding of substrate D in the absence of substrate D' to the sites that are far from fibrillarin does not lead to product D. In vivo, the sRNP is probably recycled by removal of all products after modification of all four sites. Based on our data we propose that rRNA methylation by the sR26 guide RNA occurs in a sequential manner, providing a novel mechanism to regulate correct establishment of rRNA methylation patterns. Although the role of sequential methylation remains to be confirmed for other guide sRNAs, it is tempting to speculate that a regulated order of methylation at different rRNA sites may offer elegant means to control the pathway of rRNA folding, a complex process that in part takes place contemporarily to pre-rRNA modification.

Lapinaite et al (2013) The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature, [in press, doi:10.1038/nature12581].