10.5 Processing of RNA
Post-transcriptional modifications of rRNA and tRNA will be topics of Chapter 11 as their structure and function in protein synthesis will be a focal point. Thus, this section will focus on post-transcriptional modifications of mRNA.
Prokaryotic RNA Processing
Bacterial cells do not have extensive post-transcriptional modification of mRNA primarily because transcription and translation are coupled processes. Bacterial cells lack the physical barrier of a nucleus, which allows transcription and translation machineries to function at the same time, enabling the concurrent translation of an mRNA while it is being transcribed (Fig 10.19). Within this system the NusG protein plays a critical role. NusG has three separate domains and the functions of two of them are known. The NusG N-terminal domain (NusG-NTD) has the capacity to bind to RNAP, whereas the C-terminal domain (NusG-CTD) can combine with the NusE (RpsJ) component of ribosomes. These two functions of NusG enable transcription to be coupled with translation. NusG CTD can also bind to Rho to terminate transcription (Figure 10.19).
Fig. 10.19. The roles of NusG in transcription/translation coupling. (a) Composition of an active RNAP complex. RNAP is shown in dark grey, DNA in blue and nascent RNA in red. The ribosome is shown in green with the nascent polypeptide chain in light grey; the bulge in the small subunit denotes the location of NusE (RpsJ). NusG is shown in orange: its shape denotes two functional sections. The larger section denotes the N-terminal domain, which binds to RNAP. The smaller section denotes the C-terminal domain, which interacts with NusE in situ. Rho is shown in purple. (b) After translation is completed NusG remains bound to RNAP and may also bind to Rho through the C-terminal domain leading to termination of transcription.
Eukaryotic RNA Processing
In multicellular organisms almost every cell contains the same genome, yet complex spatial and temporal diversity is observed in gene transcripts. This is achieved through multiple levels of processing leading from gene to protein, of which RNA processing is an essential stage. Following transcription of a gene by RNA polymerases to produce a primary mRNA transcript, further processing is required to produce a stable and functional mature RNA product. This involves various processing steps including RNA cleavage at specific sites, intron removal, called splicing, which substantially increase the transcript repertoire, and the addition of a 5'CAP. Another crucial feature of the RNA processing of most genes is the generation of 3′ ends through an initial endonucleolytic cleavage, followed in most cases by the addition of a poly(A) tail, a process termed 3′ end cleavage and polyadenylation (CPA).
As seen in Section 10.4, polyadenylation is a required step for the correct termination of nearly all mRNA transcripts. With the exception of replication dependent histone genes, metazoan protein encoding mRNAs contain a uniform 3' end consisting of a stretch of adenosines. In addition to deterimining the correct transcript length at transcription termination, the poly(A) tail helps to ensure the translocation of the nascent RNA molecule from the nucleus to the cytoplasm, enhances translation efficiency, acts as a signal feature for RNA degradation, and thereby contributes to the production efficiency of a protein.
CPA is carried out by a multi-subunit 3′ end processing complex, which involves over 80 trans-acting proteins, comprised of four core protein subcomplexes (Figure 10.20 A). These consist of (1) cleavage and polyadenylation specificity factor (CPSF), comprised of proteins CPSF1-4, factor interacting with PAPOLA and CPSF1 (FIP1L1), and WD repeat domain 33 (WDR33) (shown in green on Figure 10.20 A); (2) cleavage stimulation factor (CstF), a trimer of CSTF1-3 (shown in red on Figure 10.20 A; (3) cleavage factor I (CFI), a tetramer of two small nudix hydrolase 21 (NUDT21) subunits, and two large subunits of CPSF7 and/or CPSF6 (shown in orange in Figure 10.20 A); and (4) cleavage factor II (CFII), composed of cleavage factor polyribonucleotide kinase subunit 1 (CLP1) and PCF11 cleavage and polyadenylation factor subunit (PCF11) (shown in yellow on Figure 10.20 A). Additional factors include symplekin, the poly(A) polymerase (PAP), and the nuclear poly(A) binding proteins such as poly(A) binding protein nuclear 1 (PABPN1).
CPA is initiated by this complex recognising specific cis-element sequences within the nascent pre-mRNA transcripts termed polyadenylation signals (PAS). The PAS sequence normally consists of either a canonical AATAAA hexamer, or a close variant usually differing by a single nucleotide (e.g., ATTAAA, TATAAA). It is located 10 to 35 nucleotides upstream of the cleavage site (CS) usually consisting a CA dinucleotide. The PAS is also determined by surrounding auxiliary elements, such as upstream U-rich elements (USE), or downstream U-rich and GU-rich elements and G-rich sequences (DSE).
As soon as the nascent RNA molecule emerges from RNA polymerase II (RNA Pol II), the CPSF complex is recruited to the PAS hexamer through numerous interactions. Upon successful assembly of this macromolecular machinery, CPSF3 performs the endonucleolytic cleavage followed by a non-templated addition of approximately 50-100 A residues.
Figure 10.20. The core 3′ end RNA processing machinery and impact on alternative polyadenylation. (A) The core 3′ end processing machinery consists of complexes composed of multiple trans acting proteins interacting with RNA via multiple cis-elements (USE = upstream sequence element; PAS = poly(A) signal; CS = cleavage site; DSE = downstream sequence element; CTD = C-terminal domain). Upon co-transcriptional assembly of these complexes, RNA cleavage and polyadenylation occurs to form the 3′ end of the nascent RNA molecule. (B) More than 70% of all genes harbour more than one polyadenylation signal (PAS). This gives rise to transcript isoforms differing at the mRNA 3′ end. While alternative polyadenylation (APA) in 3′UTR changes the properties of the mRNA (stability, localisation, translation), internal PAS usage (in introns or the coding sequence (CDS)) changes the C-termini of the encoded protein, resulting in different functional or regulatory properties.
Figure from: Nourse, J., et. al. (2020) Biomolecules 10(6):915
Alternative polyadenylation (APA) occurs when more than one PAS is present within a pre-mRNA and provides an additional level of complexity in CPA-mediated RNA processing (Figure 10.20 B). Early studies revealed a significant portion of genes undergo APA, and with the advent of next-generation RNA sequencing technologies the large scale regulation of genes has become apparent, with approximately 70% of the transcriptome exhibiting APA regulation. As APA determines 3′UTR content and thus the regulatory features available to the mRNA, changes in the APA profile of a gene can have enormous impacts on expression.
In eukaryotes, the 5′ cap, found on the 5′ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage (Fig. 10.21). This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. It is referred to as a 7-methylguanylate cap, abbreviated m7G.
In multicellular eukaryotes and some viruses, further modifications exist, including the methylation of the 2′ hydroxy-groups of the first 2 ribose sugars of the 5′ end of the mRNA. Cap-1 has a methylated 2′-hydroxy group on the first ribose sugar, while cap-2 has methylated 2′-hydroxy groups on the first two ribose sugars. The 5′ cap is chemically similar to the 3′ end of an RNA molecule (the 5′ carbon of the cap ribose is bonded, and the 3′-OH unbonded). This provides significant resistance to 5′ exonucleases.
snRNAs contain unique 5′-caps. Sm-class snRNAs are found with 5′-trimethylguanosine caps, while Lsm-class snRNAs are found with 5′-monomethylphosphate caps. In bacteria, and potentially also in higher organisms, some RNAs are capped with NAD+, NADH, or 3′-dephospho-coenzyme A. In all organisms, mRNA molecules can be decapped in a process known as messenger RNA decapping.
For capping with 7-methylguanylate, the capping enzyme complex (CEC) binds to RNA polymerase II before transcription starts. As soon as the 5′ end of the new transcript emerges from RNA polymerase II, the CEC carries out the capping process (this kind of mechanism ensures capping, as with polyadenylation). The enzymes for capping can only bind to RNA polymerase II that is engaging in mRNA transcription, ensuring specificity of the m7G cap almost entirely to mRNA.
Figure 10.21 Structure of the 7-methylguanylate CAP.
Figure from: Brisbane
The 5′ cap has four main functions:
- Regulation of nuclear export
- Prevention of degradation by exonucleases
- Promotion of translation (see ribosome and translation)
- Promotion of 5′ proximal intron excision
In addition to the polyA tail, nuclear export of RNA is regulated by the cap binding complex (CBC), which binds to 7-methylguanylate-capped RNA (Fig 10.22). The CBC is then recognized by the nuclear pore complex and the mRNA exported. Once in the cytoplasm after the pioneer round of translation, the CBC is replaced by the translation factors eIF4E and eIF4G of the eIF4F complex. This complex is then recognized by other translation initiation machinery including the ribosome, aiding in translation efficiency.
Capping with 7-methylguanylate prevents 5′ degradation in two ways. First, degradation of the mRNA by 5′ exonucleases is prevented by functionally looking like a 3′ end. Second, the CBC and eIF4E/eIF4G block the access of decapping enzymes to the cap. This increases the half-life of the mRNA, essential in eukaryotes as the export and translation processes take significant time.
The mechanism that promotes the 5′ proximal intron excision during splicing is not well understood, but the 7-methylguanylate cap appears to loop around and interact with the spliceosome, potentially playing a role in the splicing process.
Decapping of a 7-methylguanylate-capped mRNA is catalyzed by the decapping complex made up of at least Dcp1 and Dcp2, which must compete with eIF4E to bind the cap. Thus the 7-methylguanylate cap is a marker of an actively translating mRNA and is used by cells to regulate mRNA half-lives in response to new stimuli. During the decay process, mRNAs may be sent to P-bodies. P-bodies are granular foci within the cytoplasm that contain high levels of exonuclease activity.
Figure 10.22 Importance of the 5-CAP during the lifespan of a mRNA Transcript (a) CBC is required for pre-mRNA processing. The co-transcriptional binding of CBC to 7mG prevents the decapping activities of pre-mRNA degradation complexes [DXO (decapping exoribonuclease) and Dcp (decapping mRNA) Xrn2 (5′–3′ exoribonuclease 2)] and promotes pre-mRNA processing. CBC recruits P-TEFb [Cdk9/Cyclin T1 (CycT1)] to transcription initiation sites of specific genes promoting phosphorylation of the RNA pol II CTD at Ser2 residues. This results in the recruitment of splicing factors including SRSF1, which regulates both constitutive and alternative splicing events. Furthermore, CBC interacts with splicing machinery components that results in the spliceosomal assembly. CBC interacts with NELF and promotes pre-mRNA processing of replication-dependent histone transcripts. (b) CBC forms a complex with Ars2 and promotes miRNA biogenesis by mediating pri-miRNA processing. (c) CBC/Ars2 promotes pre-mRNA processing of replication-dependent histone transcripts. (d) CBC promotes export of U snRNA. CBC interacts with PHAX, which recruits export factors including CRM1 and RAN·GTP. (e) CBC promotes export of mRNA. For export of transcripts over 300 nucleotides, hnRNP C interacts with CBC and inhibits the interaction between CBC and PHAX, allowing the CBC to interact with TREX and the transcript to be translocated to the cytoplasm. CBC interacts with the PARN deadenylase and inhibits its activity, protecting mRNAs from degradation. (f) CBC mediates the pioneer round of translation. Cbp80 interacts with CTIF, which recruits the 40S ribosomal subunit via eIF3 to the 5′ end of the mRNA for translation initiation. Upon binding of importin-β (Imp-β) to importin-α (Imp-α), mRNA is released from CBC and binds to eIF4E for the initiation of the standard mode of translation. CBC-bound mRNP components not found in eIF4E-bound mRNPs are CTIF, exon junction complex (EJC) and PABPN1. (g) The standard mode of translation is mediated by eIF4E cap-binding protein. eIF4E is a component of the eIF4F complex which promotes translation initiation.
Eukaryotic genes that encode polypeptides are composed of coding sequences called exons (ex-on signifies that they are expressed) and intervening sequences called introns (int-ron denotes their intervening role). Transcribed RNA sequences corresponding to introns do not encode regions of the functional polypeptide and are removed from the pre-mRNA during processing. It is essential that all of the intron-encoded RNA sequences are completely and precisely removed from a pre-mRNA before protein synthesis so that the exon-encoded RNA sequences are properly joined together to code for a functional polypeptide. If the process errs by even a single nucleotide, the sequences of the rejoined exons would be shifted, and the resulting polypeptide would be nonfunctional. The process of removing intron-encoded RNA sequences and reconnecting those encoded by exons is called RNA splicing. Intron-encoded RNA sequences are removed from the pre-RNA while it is still in the nucleus. Although they are not translated, introns appear to have various functions, including gene regulation and mRNA transport. On completion of these modifications, the mature transcript, the mRNA that encodes a polypeptide, is transported out of the nucleus, destined for the cytoplasm for translation. Introns can be spliced out differently, resulting in various exons being included or excluded from the final mRNA product. This process is known as alternative splicing. The advantage of alternative splicing is that different types of mRNA transcripts can be generated, all derived from the same DNA sequence. In recent years, it has been shown that some archaea also have the ability to splice their pre-mRNA.
The splicing reaction is catalyzed by the spliceosome, a macromolecular complex formed by five small nuclear ribonucleoproteins (snRNPs), termed U1, U2, U4, U5, and U6, and approximately 200 proteins (Fig. 10.23). The assembly of the spliceosome on pre-mRNA includes the binding of U1 snRNP, U2 snRNP, the pre-formed U4/U6-U5 triple snRNP, and the Prp19 complex. This assembly occurs through the recognition of several sequence elements on the pre-mRNA that define the exon/intron boundaries, which include the 5′ and 3′ splice sites (SS), the associated 3′ sequences for intron excision, the polypyrimidine (Py) tract, and the branch point sequence (BPS). The assembly of the spliceosome during the process is depicted in Figure 10.23.
Figure 10.23 Schematic representation of the spliceosome assembly and pre-mRNA splicing. In the first step of the splicing process, the 5′ splice site (GU, 5′ SS) is bound by the U1 snRNP, and the splicing factors SF1/BBP and U2AF cooperatively recognize the branch point sequence (BPS), the polypyrimidine (Py) tract, and the 3′ splice site (AG, 3′ SS) to assemble complex E. The binding of the U2 snRNP to the BPS results in the pre-spliceosomal complex A. Subsequent steps lead to the binding of the U4/U5–U6 tri-snRNP and the formation of complex B. Complex C is assembled after rearrangements that detach the U1 and U4 snRNPs to generate complex B*. Complex C is responsible for the two transesterification reactions at the SS. Additional rearrangements result in the excision of the intron, which is removed as a lariat RNA, and ligation of the exons. The U2, U5, and U6 snRNPs are then released from the complex and recycled for subsequent rounds of splicing.
Figure from: Suñé-Pou, M., et. al. (2017) Genes 8(3):87
In mammals, the first catalytic step of the splicing reaction begins when the U1 snRNP binds the 5′ SS of the intron (defined by the consensus sequence AGGURAGU), and the splicing factors SF1 and U2AF cooperatively recognize the BPS, Py, and 3′ SS to assembled complex E or the commitment complex (Figure 10.23). Subsequently, U2 snRNP and additional proteins are recruited to the pre-mRNA BPS to form the pre-spliceosome or complex A. The binding of the U4/U6-U5 tri-snRNP forms the pre-catalytic spliceosome or complex B. After RNA-RNA and RNA-protein rearrangements at the heart of the spliceosome, U1 and U4 are released to form the activated complex B or complex B* This complex is responsible for executing the first catalytic step, through which the phosphodiester bond at the 5′ SS of the intron is modified by the 2′-hydroxyl of an adenosine of the BPS to form a free 5′ exon and a branched intron (Fig. 10.24). The reaction of the 2'-hydroxyl from the branchpoint adenosine nucleotide is known as a transesterification reaction. During this process, additional rearrangements occur to generate the catalytic spliceosome or complex C (Fig. 10.23), which is responsible for catalyzing the second transesterification reaction leading to intron excision and exon–exon ligation (Fig. 10.24). The resulting intron structure is referred to as a lariat structure. After the second catalytic step, the U2, U5, and U6 snRNPs are released from the post-spliceosomal complex and recycled for additional rounds of splicing.
Figure 10.24 Transesterification Reactions Involved in mRNA Splicing. (A) Schematic diagram of the pre-mRNA with exons and introns indicated. Key sequences are required for splicing at the 5' and 3' intron locations, and for the recognition and positioning of the branchpoint Adenosine residue for the first transesterification reaction. (B) Schematic of the two transesterification reactions required for intron removal. The branchpoint 2'-OH residue mediates attack on the 5'-phosphate of the intron guanosine residue located at the 5'-splice site. This releases the 3' hydroxyl of Exon 1 which subsequently mediates attack of the 5' phosphate of the first guanosine residue in Exon 2. The 3' hydroxyl of the intron guanine residue is released forming the Lariat structure and Exon 1 is ligated to Exon 2.
Alternative Splicing (AS) offers an additional mechanism for regulating protein production and function. AS options are determined by the expression of or exposure to in trans elements present within unique cellular locations and environments. Additional sequence elements within the mRNA, known as exonic and intronic splicing silencers or enhancers (ESS, ISS, ESE, and ISE, respectively), participate in the regulation of AS. Specific RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins, recognize these sequences to positively or negatively regulate AS (Figure 10.25). These regulators, together with an ever-increasing number of additional auxiliary factors, provide the basis for the specificity of this pre-mRNA processing event in different cellular locations within the body.
Figure 10.25 Alternative splicing (AS) regulation by cis mRNA elements and trans-acting factors. The core cis sequence elements that define the exon/intron boundaries (5′ and 3′ splice sites (SS), GU-AG, polypyrimidine (Py) tract, and branch point sequence (BPS)) are poorly conserved. Additional enhancer and silencer elements in exons and in introns (ESE: exonic splicing enhancers; ESI: exonic splicing silencers; ISE: intronic splicing enhancers; ISI: intronic splicing silencers) contribute to the specificity of AS regulation. Trans-acting splicing factors, such as SR family proteins and heterogeneous nuclear ribonucleoprotein particles (hnRNPs), bind to enhancers and silencers and interact with spliceosomal components. In general, SR proteins bound to enhancers facilitate exon definition, and hnRNPs inhibit this process. These trans-acting elements are expressed differentially within different locations or under different environmental stimuli to regulate AS.
Figure from: Suñé-Pou, M., et. al. (2017) Genes 8(3):87
There are several different types of AS events, which can be classified into four main subgroups. The first type is exon skipping, which is the major AS event in higher eukaryotes. In this type of event, a cassette exon is removed from the pre-mRNA (Fig. 10.26 a). The second and third types are alternative 3′ and 5′ SS selection (Fig. 10.26 b & c). These types of AS events occur when the spliceosome recognizes two or more splice sites at one end of an exon. The fourth type is intron retention (Fig. 10.26 d), in which an intron remains in the mature mRNA transcript. This AS event is much more common in plants, fungi and protozoa than in vertebrates. Other events that affect the transcript isoform outcome include mutually exclusive exons (Fig. 10.26 e), alternative promoter usage (Fig. 10.26 f), and alternative polyadenylation (Fig. 10.26 g).
Figure 10.26 Schematic representation of different types of alternative transcriptional or splicing events, with exons (boxes) and introns (lines). Constitutive exons are shown in green and alternatively spliced exons in purple. Dashed lines indicate the AS event. Exon skipping (a); alternative 3′ (b) and 5′ SS selection (c); intron retention (d); mutually exclusive exons (e); alternative promoter usage (f); and alternative polyadenylation (g) events are shown. Like alternative splicing (AS), usage of alternative promoter and polyadenylation sites allow a single gene to encode multiple mRNA transcripts.
Figure from: Suñé-Pou, M., et. al. (2017) Genes 8(3):87