Co‐transcriptional assembly mechanisms of protein‐RNA complexes

This graphical review provides a mechanistic overview of different molecular processes that are tightly coupled and cooperate to achieve efficient and spatial‐temporally regulated co‐transcriptional protein‐RNA complex assembly, including co‐transcriptional RNA folding, processing, modification and the assembly in context of biomolecular condensates.

The assembly and disassembly of protein-RNA complexes (ribonucleoprotein, RNP) is one of the most fundamental molecular processes in all life forms, underlying transcription, translation and splicing, and errors in RNP assembly underlie many human diseases [1].The formation of an RNP complex involves synthesis and correct folding of the individual protein and nucleic acid components and formation of specific intermolecular interactions between them.RNPs start to form as soon as the nascent RNA emerges from the RNA polymerase (RNAP) [2,3].
The context in which RNA folds co-transcriptionally is very complex and is modulated by many different factors: i) Proteins and other ligands (non-coding RNAs, small molecules such as metabolites, antibiotics or drugs) can bind, either transiently or stably, to the nascent RNA, thereby influencing the RNA folding pathway by either refolding the RNA or by transiently sequestering RNA segments [4,5].ii) RNA modification alters nucleotide chemistry and modification enzyme binding can alter nascent RNA folding [6].iii) Co-transcriptional RNA processing generates new RNA molecules, which become separated from the RNA synthesizing machinery and therefore, are not dependent on the co-transcriptional folding constraints anymore [7].iv) Within a cellular context, RNPs may not assemble in isolation but multiple RNPs can interact with each other, sometimes leading to phase separation [8].Overall, the formation of an RNP complex is a very complicated process involving a multitude of factors, which are tightly coupled and must be optimally coordinated to achieve efficient co-transcriptional RNA folding and RNP assembly.

Co-transcriptional RNA folding and RNP assembly
RNA folds co-transcriptionally while still being synthesized by the RNAP [2,3].In contrast to proteins, RNA is conformationally much more dynamic and can also form alternative non-native structures with a similar free energy as the native structure [9].A complex but well-studied example of RNP assembly is bacterial co-transcriptional ribosome assembly [10][11][12].Co-transcriptional rRNA folding is dependent on the transcription rate [13,14] and is assisted by both the ribosomal proteins (r-proteins) and over 30 ribosome assembly factors.These together remodel the architecture of the RNP assembly intermediates by inducing conformational rearrangements in the rRNA and support the search for its native fold [12,[14][15][16].As the rRNA folding proceeds, the r-proteins get stably incorporated into the emerging ribosome, whereas assembly factors get released after successful assembly of specific intermediates structures, providing a means for quality control [11,12,14,15,17].Overall, the r-proteins and assembly factors together smoothen the rugged RNA folding landscape by destabilizing misfolded, non-functional states and stabilizing transition states [4,5].

RNA processing
Most types of RNAs are processed into their mature forms from long precursor RNAs providing opportunities for regulation and quality control [7].Several processing steps, such as capping, splicing, modification and polyadenylation of eukaryotic mRNA, occur during transcription and involve the assembly of large protein or RNP complexes onto the nascent RNA and the transcription machinery [7].Correct processing can require that sites far away in sequence have to be brought together once both sites have been transcribed.Thus, nature has developed strategies that keep the 5′ end in close proximity to the emerging transcript, by looping-out the growing RNA chain, until it can form a helix with its complementary target site at the 3′-end.For example, in bacterial rRNA transcription, the rRNA transcription antitermination complex (rrnTAC) simultaneously binds to the 5′-end of the rRNA and the RNAP and thereby keeps the 5′-end and the 3′-end of the rRNA in close proximity for efficient processing by RNase III to release the initial 16S rRNA precursor from the single rRNA transcript [18].In case of the spliceosome, retaining proximity of the 5'-splice site (SS) and 3'SS during transcription is likely accomplished by the U1 small nuclear ribonucleoprotein that simultaneously associates with the 5'-SS and RNA polymerase II [19].

RNA modification
Nucleotide modifications expand the repertoire for molecular interactions of an RNA molecule by changing the chemistry and therefore, they are often used by organisms to tune RNA folding, its interaction with proteins and therefore functionality [6].Out of possible >150 RNA modifications, the N 6 -methyladenosine (m 6 A) modification is the most prevalent one in eukaryotes.It is often co-transcriptionally introduced by the m 6 A writer protein-complex METTL3/METTL14, can specifically be recognized by different m 6 A reader proteins (e.g.YTHDC1) in a context-dependent manner and can be dynamically removed by m 6 A eraser proteins (e.g.FTO and ALKBH5) [20].m 6 A can dynamically reshape RNA secondary structure by weaking A-U base-pairs, which in turn can create new binding sites for proteins (e.g.hnRNPC), therefore altering RNP assembly on the modified RNA target [21].

Small molecule -RNA interactions
Specific RNA structures, termed riboswitches, can bind small molecules resulting in functional structural changes [22].They are highly structured regulatory RNAs often located in 5′-UTRs of mostly bacterial mRNAs and sense ions or small molecules with high specificity, thereby modulating downstream gene expression at several levels (transcription, translation, mRNA splicing and decay) [23].The sensed metabolite is typically part of an enzymatic pathway involving the regulated gene product and induces a switch to alternative RNA structures upon binding to change gene expression.For example, for the translational lysC riboswitch, lysine binding not only shuts down translation by occlusion of the ribosome binding site but also initiates mRNA decay by releasing RNase E binding sites, thereby making gene repression more robust [24].

Compartmentalization and biomolecular condensates
Cells organize many processes in distinct compartments (organelles) that are physically separated by membranes.Compartmentalization can also be achieved by phase separation out of solution resulting into biomolecular condensates such as P-bodies (RNA processing and storage) or the nucleolus (ribosome assembly) [8].Some complex RNPs require the transitioning between different cellular compartments for their successful assembly.For example, eukaryotic rRNA transcription, processing, modification and early rRNP assembly are part of a maturation gradient established along the different liquid phases of the multilayered nucleolus before further ribosome assembly proceeds in the nucleoplasm and finalizes in the cytoplasm [25,26].Phase separation can be driven by active processes such transcription (e.g. for nucleolus [26] or paraspeckles [27]).Driving forces for phase separation involve the formation of fast-exchanging RNA-RNA, protein-protein, protein-RNA interactions aided by multidomain proteins that allow the formation of a multivalent and dynamic network of molecular interactions by using both intrinsically disordered regions and folded domains (e.g.RRM, KH, SH3), with post-translational modifications (PTMs) modulating this process [28,29].
.g. CspA, r-proteins) transiently bind and assist early RNA folding co-transcriptional folding of long-range RNA-RNA interactions is difficult initial transient binding of early r-proteins shapes their binding Authors.FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.doi -10.1002/1873-3468.14758