THE RNA INFRASTRUCTURE: An Introduction to ncRNA Networks

The RNA infrastructure connects RNA-based functions. With transcription- to-translation processing forming the core of the network, we can visualise how RNA-based regulation, cleavage and modification are the backbone of cellular function. The key to interpreting the RNA-infrastructure is in understanding how core RNAs (tRNA, mRNA and rRNA) and other ncRNAs operate in a spatial-temporal manner, moving around the nucleus, cytoplasm and organelles during processing, or in response to environmental cues. This chapter summarises the concept of the RNA-infrastructure, and highlights examples of RNA-based networking within prokaryotes and eukaryotes. It describes how transcription-to-translation processes are tightly connected, and explores some similarities and differences between prokaryotic and eukaryotic RNA networking.

INTRODUCTION

RNA biology in both eukaryotes and prokaryotes exists in a spatiotemporal network of processes we call the RNA-infrastructure. In eukaryotes, there are numerous subtypes of noncoding (nc) RNA genes involved including rRNA, mRNA, tRNA, snRNA, snoRNAs, several classes of regulatory RNAs (RNAi) and many long ncRNAs. In prokaryotes, in addition to tRNAs, mRNAs and rRNAs, we can have small RNAs, CRISPRs and tmRNAs, and even viruses can contain small RNAs. ncRNAs are generally involved in the transcription-to-translation processes surrounding the conversion and regulation of information from DNA to protein, implicated in viral defence mechanisms, or are involved in gene regulation (e.g., RNA interference; RNAi). What we are only beginning to understand is how these processes are integrated, and how RNA plays a previously understated role in the overall regulation of the cell.

There are some key differences in cells that are differentiated (i.e., from multicellular eukaryotes), single celled eukaryotes and prokaryotes, but there are also striking similarities in how RNA processing and regulation works in different types of cells, giving us clues to their evolution. Although we are more familiar with RNA networks from eukaryotes, prokaryotic noncoding RNA research is using concepts developed from eukaryotic work to discover new RNA-based systems in bacteria and archaea. Although finding RNA genes is becoming a standard step in genomic investigations, it is now clear that discovering connections between these genes, and their associated proteins is just as important. Once we add in regulatory and epigenetic elements (such as methylation and histone modification) our regulatory networks can become very complex, but these complex networks have the ability to indicate linkages between cellular machineries not previously observed. The examples in this chapter will show how RNA-based processes within both prokaryotic and eukaryotic cells interact in networks in both a spatial and temporal manner.

RNAs PROCESSING OTHER RNAs

A good example in how RNA-processes are connected comes from examining the transcription-to-translation processes which form the core of the RNA-infrastructure (Fig. 1).¹ The processing of the three core-RNAs in eukaryotes (mRNA, tRNA and rRNA) includes the RNA-based mechanisms of RNA cleavage and modification (Fig. 1A). In euaryotes these are: rRNA by RNase MRP and snoRNAs; tRNA by RNaseP; and mRNAs spliced by snRNAs within the spliceosome. We can then expand this idea to include spatial movement and regulation during RNA-processing (Fig. 1B). In prokaryotes we still have tRNAs and rRNAs being processed either directly by RNAs (e.g., RNase P of tRNA and tmRNA) or indirectly (where rRNAs are released by tRNA processing) (Fig. 1C).

Examining the connections between these processes in more detail we see networking between different mRNA machineries. For example, transcription by RNA Polymerase II (Pol II) and mRNA splicing in mammals are carried out in close proximity,² and this coupling may protect the newly synthesised RNA from degradation³ before the termination of transcription.^4,5 Some splicing may occur cotransciptionally and this significantly improves processing efficiency (reviewed in ref. 6). At the other end of the transcript, 3′-end cleavage and polyadenylation of mRNA can be promoted by splicing proteins (e.g., U2AF65 reviewed in ref. 7). It is clear that splicing (the processing of mRNA with snRNAs) connects to other mRNA-processes including RNA localisation, translational yield and mRNA decay.⁸ In another example, the Exon Junction Complex (EJC) is a set of proteins deposited on 5′ end of the exon during the second step of the splicing cycle, and remain bound to the spliced mRNA as it is exported to the cytoplasm.⁹ This complex interacts transiently with many factors that connect the mRNA to the downstream RNA processing network,^10,11 as it is a major link between mRNA-splicing and mRNA export, as well as having a potential role in RNA degradation. The EJC appears to relay the previous location of introns,⁸ and thus detects incorrect splicing that introduces premature stop-codons. It has been shown that in mammals at least, spliceosomal proteins and especially those involved in exon-definition, remain associated with the pre-mRNA to be available for the splicing of the next introns, thus allowing for efficient splice site recognition for subsequent introns since splice site recognition only needs to be carried out once for a site.⁶ With splicing central to downstream RNA processing it is not surprising that many proteins are now seen as having roles in splicing as well as their own function (e.g., transcription or capping). However, it remains to be seen whether these proteins actually influence catalysis in the spliceosome, or are detected due to the close proximity of these RNA processing complexes.

Similarly, transcribed pre-tRNAs require processing before being able to function as amino acid transfer molecules for translation. Leader sequences at the 5′ and 3′ ends of the pre-tRNAs require cleaving, introns within the tRNA may need to be removed and in some cases a 3′ CCA tail needs to be added.¹² In addition, certain nucleotides within the tRNA require modification by aminoacylation. The ribonucleoprotein RNase P is responsible for cleaving the 5′ leader sequence of pre-tRNAs in all cells, although the overall structure of this protein-RNA complex differs in eukaryotes, bacteria and archaea. In bacteria there is one small protein that plays diverse roles such as enhancing substrate binding, altering substrate recognition, stabilising RNA conformation, and aiding catalysis by discriminating between the substrate and product by binding to the 5′ leader sequence of the pre-tRNA.^13,14 Eukaryotes have 9-10 proteins in the complex with a single RNA. Archaeal RNase P also has multiple proteins (five including the ribosomal protein L7Ae) which do show some homology to some of the eukaryotic RNase P proteins. The RNase P RNA from some representatives from each kingdom can be induced to perform weak catalysis without its accompanying proteins, but only with high salt and high cation conditions in vitro (summarised in ref. 15).

RNase P plays key networking roles in both the eukaryote's and prokaryote's RNA infrastructure, resulting in the cleavage of additional substrates and the repression of transcription (Fig. 2).^16,17 In bacteria, as well as cleaving the 5′ leader sequence of tRNAs, it cleaves a similar leader sequence for tmRNA. tmRNA (transfer-messenger RNA) is a specialised tRNA molecule that together with the SmpB protein (small protein B) rescues stalled ribosomes in a process called trans-translation (reviewed in ref. 18).With a structure partly a tRNA molecule and partly an mRNA molecule,¹⁹ the tRNA part binds to the stalled ribosome, allows the translation to proceed along the mRNA part which encodes a distinctive degradation signal and a translation stop signal. When the mistranslated protein is released after the stop signal it is targeted for degradation. This process of trans-translation is conserved throughout bacteria and is also present in some mitochondria and chloroplasts.^20,21 In prokaryotes other cleavage products by RNase P include some riboswitches and some viral RNAs as well as the 4.5S rRNA which is part of the Signal Recognition Particle involved in post-translational transport (reviewed in ref. 22).The eukaryotic counterpart of the 4.5S rRNA (7SL RNA) is also cleaved by RNase P (reviewed in ref. 13). In yeast the HRA1RNA and some C/D box snoRNAs are processed by RNase P although whether cleavage is the exact mechanism is yet to be completely determined. In humans MALAT1, another long ncRNA, is cleaved by RNase P. tRNAs in organelles within eukaryotes (in some species) either encode their own RNase P RNA (e.g., the yeast S. cerevisiae), use the nuclear counterpart (e.g., humans) or occasionally do without the RNA component altogether.²³ Additionally, in the archaeans Nanoarchaeum equitans and Pyrobaculum aerophilum and the hyperthermophilic bacterium Aquifex aeolicus, there does not appear to be any RNase P-like RNA sequence in their genomes.²⁴ In N. equitans the requirement for RNase P has been replaced by a strict placement of the promoter 26 nucleotides upstream of the mature tRNA sequence allowing transcription of leaderless tRNAs.²⁵

There is a feedback affect of RNase P on its own polymerase RNA Pol III¹⁶ and the polymerase affecting rRNA transcription, RNA Pol I.¹⁷ Thus, RNase P in eukaryotes has a large effect on other aspects of RNA processing including splicing (U6 snRNA is transcribed by RNA Pol III), and RNA modification (by some yeast C/D box snoRNAs). It clearly plays a central role in the RNA infrastructure of both eukaryotes and prokaryotes and it is likely that other substrates and processing connections, especially in prokaryotes, are still to be uncovered.

RNase MRP is a ribonucleoprotein found only in eukaryotes, but closely related and sharing many of the same proteins with RNase P (for a review see ref. 13). It too has multiple roles (Fig. 3), processing the A3 site of rRNA in the nucleolus, a critical cell cycle control protein (Cyclin B2) in the cytoplasm, and the D-loop of mitochondrial DNA (MtDNA) in the mitochondria to generate RNA primers for Mt DNA replication. This is a good illustration of the spatial nature of RNA-Protein complexes that have different roles in different cellular compartments. RNase MRP is transcribed by RNA Pol III and thus is affected by the RNase P feedback on the polymerase.

Other aspects of rRNA processing in eukaryotes are linked to transcription and downstream rRNA maturation. Extensive modification of the pre-rRNAs includes methylation of 2′ hydroxyl groups of ribose (guided by C/D box snoRNAs [small nucleolar RNAs]) and pseudouridine formation from uracil (guided by H/ACA snoRNAs).²⁶ In vertebrates, these snoRNAs are mostly found within introns, and are spliced out by snRNAs, illustrating the strong network of RNA biogenesis and splicing machineries. Yeast models (primarily in S. cerevisiae) indicate that RNA Pol I, elongation factors and rRNA sequence elements appear to optimize transcription elongation and co-ordinate interactions (including those with snoRNAs) with the pre-rRNA for correct rRNA processing and ribosome assembly.²⁷ In addition, a protein complex of three transcription factors (the CURI complex comprising of Rap1, Fhl1 and Ifh1) links ribosomal protein production and pre-rRNA processing.²⁸ Thus, rRNA processing also uses feedback from the later stages of processing to regulate transcription.

SPATIAL REGULATION OF EUKARYOTIC RNA PROCESSING

Spatial placement of both RNA and protein macromolecular components plays an important part in the regulation of RNA-processing. In eukaryotes, this is clearly demonstrated by how RNAs move through nuclear bodies (such as Cajal bodies, Gems and nucleoli) and for some of them, into cytoplasmic bodies such as P-bodies and RNA granules. As an example, Figure 4 illustrates the biogenesis of snRNAs and snoRNAs in humans. Typically there are different stages of RNA-processing taking place within different nuclear sub-compartments, but for the Sm-class-snRNAs, the processing moves to the cytoplasm, before the re-import of the snRNP-complexes back to the nucleus. In contrast, the Lsm-class snRNAs (U6 and U6_atac snRNAs) in humans never leave the nucleus,²⁹ although in yeast there may be some nuclear export and re-import of U6 snRNA.²⁹ Cajal Bodies in particular appear to be important sub-nuclear compartments for RNPs since they are not only repositories for the biogenesis of RNPs. Mature snRNPs travel through Cajal Bodies, sometimes moving from one Cajal body to another suggesting that the Cajal Body is being used as a 'recycling center', enabling the re-assembly of the tri-snRNPs.³⁰ In contrast, the assembly of C/D box snoRNPs appears to occur cotranscriptionally, but much of the intra-nuclear and intra-cellular trafficking of snoRNPs remain to be characterised.³¹

A feature of intra-cellular RNP trafficking is how some proteins assist these different RNPs in different manners. One such group of proteins linking snRNA and snoRNA biogenesis (Fig. 4) is the PHAX complex (consisting of PHAX, Cap Binding Protein (CBC), CRM1 and RanGTP) which in humans at least, transports snRNAs from the nucleus to the cytoplasm as well as transport of some snoRNAs (especially U3, U8, U13) around the nucleus to speckles, Cajal bodies and nucleoli.³² Although PHAX is a metazoan protein there has been a similar protein characterised in the protist Cryptosporidium parvum.³² Another important RNA-escorting macromolecule is the SMN protein complex, which is found in the nucleoplasm and nuclear bodies called Gems.³³ The SMN complex scrutinizes cellular RNAs to ensure that Sm cores (of highly reactive RNA-binding Sm proteins) are only assembled on proper snRNAs,³⁴ and the Gemin5 protein of this SMN complex can distinguish snRNAs from other cellular RNAs for snRNP biogenesis.³⁴ The SMN complex also plays a role in other biogenesis pathways including those for hnRNPs and microRNPs.³³ The above pathways for snRNP and snoRNP biogenesis have been largely characterised for mammalian and yeast systems, and although there is now some plant information,³⁵ there is little known about how these RNPs complexes form in the many different groups of protists. As with plants we expect some different proteins to be involved and there will likely be different pathways.

After nuclear export some mRNAs are translated immediately, but many mRNAs are recruited to RNA granules in the cytoplasm until developmental or environmental cues signal their translation.³⁶ Cytoplasmic RNA granules (reviewed in refs. 36,37)include Processing-bodies and Stress Granules as well as compartments found in germ cells (polar and germinal granules) and neurons (neuronal granules). Processing-bodies (P-bodies or GW bodies) are involved with post-transcriptional processes, including mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression and RNA-mediated gene silencing (reviewed in ref. 38). mRNA degradation is initiated by the deadenylation (shortening) of the 3′ polyA-tail followed by decapping.³⁹ Stress Granules are a cytoplasmic RNA granule that typically forms during stress response (whereas P-bodies are present continuously).⁴⁰ Stress Granules contain polyadenylated transcripts and are not degraded, making them available for rapid re-initiation after stress recovery,⁴⁰ whereas mRNAs recruited to P-bodies are largely deadenylated.³⁷ mRNAs within stress granules P-bodies and Stress Granules constantly exchange RNAs and proteins with the cytosol³⁷ and mRNAs can move from one to the other. P-bodies have been investigated in yeasts, plants, trypanosomatids, insects and vertebrates (reviewed in ref. 37) and thus are likely to be important eukaryotic RNA-based cellular features. Evidence is suggesting that Stress Granules are the consequence, not the cause of the shut off of translation during stress, and the formation of critical macromolecules may be linked to the sequestering of key components (reviewed in ref. 37).

Although RNAs such as miRNAs, siRNAs and tRNAs typically act in the cytoplasm, there are some miRNAs that may re-enter the nucleus, possibly playing a role in modification or nuclear component assembly processes (summarised in refs. 41,42). tRNAs in particular show interesting nuclear-cytoplasmic dynamics. tRNA transcription and 5′ processing is typically in the nucleolus, however 3′ processing has been found mostly in the nuceloplasm; tRNA modification is usually in the cytoplasm but in some species, tRNA splicing takes place on the mitochondrial cytoplasmic surface (reviewed in ref. 42). Subsequently however, a retrograde pathway exists where the tRNAs are imported from the cytoplasm back into the nucleus⁴³ but can then be re-exported to the cytoplasm in response to nutrient availability.⁴⁴ Although major studies of tRNA cellular dynamics has been mainly in yeast, the retrograde process (moving cytoplasm to nucleus) at least appears conserved in vertebrates (summarised in ref. 42).

Spatiotemporal movement of RNAs or key components of RNA-based machineries, is not restricted to eukaryotes. The gram-negative bacteria Caulobacter crescentus is dimorphic in that it has a stalked form that adheres to surfaces (with a holdfast and stalk), and a swarmer form that is mobile with a flagellum. Often used as a model for bacterial cell cycle and cell differentiation studies, C. crescentus shows substructure localisation and temporal timing of trans-translation.^45-47 tmRNA and its small protein SmpB are colocalised to a helix-like pattern in swarmer and predivisional cells but they are delocalised in stalked cells.⁴⁶ However, the protein RNase R which interacts with the tmRNA is localised separately to another helix-like pattern that is out of phase with the tmRNA-SmpB pattern. Trans-translation requires that the individual tmRNA-SmpB molecules would have to disassociate from the helix-like structure in order to pass through the ribosome, and it is feasible that these structures facilitate the regulation of trans-translation.⁴⁶ In a possible feedback mechanism, the tmRNA of C. crescentus is regulated in the cell cycle by temporally controlled transcription and translation.⁴⁵ With trans-translation required for many functions across bacteria, including sporolation in Bacillus subtilis,⁴⁸ symbiosis in Bradyrhizobium japonicum¹⁰⁹, and pathogenecity in Salmonella enteria (summarised in ref. 47), the tight regulation of the RNA component is not unexpected. It is even possible that these helix-like structures seen in C. crescentus are analogous to the P-bodies we find in eukaryotes.

RNA REGULATION, CONNECTING COMPONENTS OF THE RNA-INFRASTRUCTURE

CONCLUSION

Within the cell we see networks of RNAs involved in cleaving, modifiying or guiding the processing of other RNAs, and over time these networks have appeared to have exponentially expanded in complexity. Of course, the genes and their interactions were there all along and it is our knowledge that has grown. The modern RNA world is a tight-knit one where molecular machineries share proteins and sometimes RNAs. With the environment of the cells being so tightly packed (i.e., molecular crowding),¹⁰⁷ this sharing permits connected machineries to respond together to cellular signals. Whether universal RNAs have remained because they are intrinsically better suited for this role remains an interesting evolutionary question.

It is clear that our view of the prokaryotic modern RNA world is changing as the emphasis on the biogenesis and processing of the core RNAs (mRNA, tRNA and rRNA) has shifted now to tackle RNA-based regulation. Although prokaryotic genomes do not carry anywhere near the same degree of noncoding transcripts we see in eukaryotes, they nevertheless carry a wealth of RNA genes with which we can already see networking. As more is uncovered other interesting studies will no doubt tackle the similarities and differences between prokaryotic and eukaryotic RNA networks. From here we can investigate whether these networks evolved separately or have stemmed from an 'RNP-world' ancestor.¹⁰⁸ What is very clear is that in modern cellular networks, the role of RNA genes and how they connect in cellular networks makes them as important as their protein-coding cousins.

ACKNOWLEDGEMENTS

Many thanks go to Prof. David Penny, Prof. Mike Hendy and Prof. Pete Lockhart for stimulating discussions and support.

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Footnotes

a

In a wave of confusion we are also seeing different types of regulatory RNAs given the same prefix. Two types of small RNA, transcription-initiation RNAs,⁶⁵ and tiny RNAs ⁶⁶ have both been given the name tiRNAs. To avoid confusion the expanded name rather than tiRNA will be used for both cases.