Review
A roadmap for rRNA folding and assembly during transcription

https://doi.org/10.1016/j.tibs.2021.05.009Get rights and content

Highlights

  • Nascent RNAs form variable structures as they are transcribed, creating an unavoidable hazard for RNP assembly.

  • To form the correct structures during synthesis, strategies have emerged such as metastable RNA switches, modular domain folding, and protein-guided folding.

  • Proteins primarily form transient, non-native complexes with elongating RNAs.

  • Transient protein binding may be sufficient to guide RNA folding and initiate RNP assembly during transcription.

  • Other transcription-coupled processes, such as RNA modification, binding of assembly factors, and processing by nucleases, kinetically compete with RNA folding and the assembly of stable RNP domains.

Ribonucleoprotein (RNP) assembly typically begins during transcription when folding of the newly synthesized RNA is coupled with the recruitment of RNA-binding proteins (RBPs). Upon binding, the proteins induce structural rearrangements in the RNA that are crucial for the next steps of assembly. Focusing primarily on bacterial ribosome assembly, we discuss recent work showing that early RNA–protein interactions are more dynamic than previously supposed, and remain so, until sufficient proteins are recruited to each transcript to consolidate an entire domain of the RNP. We also review studies showing that stable assembly of an RNP competes against modification and processing of the RNA. Finally, we discuss how transcription sets the timeline for competing and cooperative RNA-RBP interactions that determine the fate of the nascent RNA. How this dance is coordinated is the focus of this review.

Section snippets

The fate of RNA begins at birth

Many RNAs fold into stable structures that allow them to perform essential biochemical processes, such as gene regulation, splicing, and protein synthesis. A classic example is the bacterial 16S rRNA that adopts a unique structure and binds 20 or more ribosomal proteins (RPs) to form the 30S ribosomal subunit that participates in protein synthesis. Early electron micrographs of actively transcribed rDNA revealed the precursor rRNA associating with proteins as it was elongated by RNA polymerase

Folding is an unavoidable hazard during RNA synthesis

Nascent transcripts begin to fold as they emerge from the elongating RNAP, because the timescale for RNA base pairing is much shorter than the timescale for transcription (Figure 1; Box 1). Consequently, interactions between nucleotides near the 5′ end of the RNA form seconds or minutes earlier than interactions with downstream nucleotides [14,15]. It has long been thought that sequential RNA folding during transcription streamlines RNP assembly by committing the RNA to a specific subset of

Conformational switching is an evolved trait

Countless noncoding RNAs require long-range interactions spanning hundreds of nucleotides. How do such RNAs fold correctly if these long-range interactions are likely to mispair during transcription? Key insights have come from studies of riboswitches, which regulate transcription termination in response to ligand binding [24]. A number of chemical footprinting, computational, and single-molecule methods have been recently deployed to understand how riboswitches fold into structures that enable

Variable RNA folding during transcription delays protein recruitment

Folding strategies that rely only on interactions within the RNA itself become increasingly less efficient for long transcripts due to the sheer number of different structures that may form. However, misfolding of long RNAs can be circumvented by RBPs, which may capture native interactions or lower the kinetic barriers to RNA refolding [28., 29., 30.]. Moreover, assembly of a multibody RNP affords greater control over the timing of molecular interactions during RNA elongation. These

Long-range RNA interactions fall into the trap

One may ask whether certain protein binding sites are more vulnerable to variable folding than others. Interestingly, the binding sites of uS4 and uS7 both straddle long-range 16S helices 3 and 28 that close the 5′ and 3′ major domains, respectively (Figure 2A,B). This raised the possibility that stable binding is prevented by mispairing of these long-range interactions during transcription. Duss and coworkers tested this idea using single-molecule Förster resonance energy transfer (smFRET) and

Unstable complexes drive assembly

If most RPs sample the rRNA dynamically as it first starts to fold during transcription, and if too-stable local interactions can trap the rRNA in unproductive intermediates, this implies that transient non-native complexes may help kick-start ribosome assembly. How can this be? Many RPs protect their binding sites in stages [13], suggesting that the protein and RNA cofold into the native complex after they come together. For example, detailed studies of uS4 binding showed that the initial

Linking transcription elongation with RNP assembly

If proper assembly of an RNP depends on metastable structures, some transcripts may only be competent to recruit partner RBPs within a ‘window of opportunity’ that is established by the timeline of RNA synthesis (Figure 4, Key figure). Evidence for the importance of transcription speed comes from the inability to assemble functional 50S subunits when T7 RNAP was used to transcribe the 23S rRNA in E. coli cells at 37°C [45]. This defect was suppressed by growing cells at a lower temperature that

Assembly factors and modification enzymes

Ribosome assembly involves many auxiliary proteins, such as AFs, modifying enzymes, and processing enzymes (Figure 4). These factors often open or distort the rRNA structure and time the establishment of long-range interactions in the immature subunits [35]. While most bacterial AFs function after transcription and initial processing of the pre-rRNA by RNase III [52], many yeast and human AFs are core components of large processomes that assemble on the pre-rRNA during transcription [53,54].

Concluding remarks

Details of how ribosome assembly is coupled with transcription and cotranscriptional RNA folding are beginning to emerge, offering lessons for the assembly of other RBPs. Since RNA folding occurs concomitantly with synthesis and is inherently heterogenous, proteins must recognize and bind to an evolving set of RNA structures during the early stages of assembly. It is becoming clear that RNAs can adopt a range of different structures during transcription, particularly when the final structure is

Acknowledgments

We thank all the members of the Woodson laboratory for helpful discussions. M.L.R and S.A.W. are funded by the National Institutes of Health (R35 GM136351-01 to S.A.W. and K99 GM140204-01 to M.L.R.).

Declaration of interests

The authors declare no competing interests.

Glossary

Assembly factor
a protein that assists the assembly or maturation of ribosomal subunits but is not a component of the mature ribosome.
Backtracking
a type of long-lived transcriptional pause involving reverse translocation of the RNA and DNA through a secondary channel within RNAP [48]. Backtracked polymerases can be reactivated through RNA cleavage with the help of transcription factors such as GreA/GreB in bacteria and TFIIS in yeast [71].
Helix junction
intersection of two or more RNA double

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