RNA Processing II: Capping & Polyadenylation

Beyond the process of splicing, eukaryotic cells perform several other distinct types of processing on RNA molecules.
mRNAs are specifically subject to two major modifications: * Capping at the 5’ end. * Polyadenylation at the 3’ end.
Capping and polyadenylation are considered essential molecular elements required for the proper biological function of mRNA.

By the year 1974, researchers discovered that mRNAs from a variety of eukaryotic species and viruses were methylated.
A significant concentration of this methylation was found clustered at the 5’-end of the mRNA molecule.
This specific cluster of methylation forms the structure currently referred to as the ‘cap’.

Early investigations into cap structure utilized viral mRNA because it was easier to purify and study than cellular mRNA.
Positioning: The cap is located at the 5’-terminus of the RNA.
Molecular Component: The cap consists of a modified guanine known as 7-methylguanosine ( $m^7G$ ).
Linkage: The linkage joining the cap to the first nucleotide is a triphosphate linkage.
Phosphate Retention: The $\beta$ -phosphate of a nucleoside triphosphate remains only in the first nucleotide of an RNA molecule.

Based on studies involving base hydrolysis of labeled viral mRNA followed by ion exchange chromatography, the following charges are contributed by the cap components: * The $m^7G$ moiety contributes one positive charge. * The triphosphate linkage contributes three negative charges. * The phosphodiester bond contributes one negative charge. * The terminal phosphate contributes two negative charges.

Cap synthesis is an early event in transcription, occurring before the RNA chain reaches a length of $30\,\text{nt}$ .
The process follows four specific enzymatic steps: 1. RNA triphosphatase: Removes the terminal phosphate from the nascent pre-mRNA. 2. Guanylyl transferase: Adds a capping GMP moiety derived from GTP. 3. Methyl transferase: Methylates the $N^7$ position of the capping guanosine. 4. Methyl transferase: Catalyses the $2’-O$ -methylation of the penultimate nucleotide (the nucleotide following the cap).

The cap structure serves at least four critical functions: 1. Protection of mRNAs from degradation, thereby increasing the half-life of the molecule. 2. Enhancement of the translatability of mRNAs. 3. Facilitation of mRNA transport out of the nucleus into the cytoplasm. 4. Enhancement of the efficiency of mRNA splicing.

Definitions: * Polyadenylation: The process of adding a poly(A) tail to RNA. * Poly(A) tail: A long chain consisting of AMP residues. * Heterogeneous nuclear mRNA (hnRNA): A precursor molecule for mature mRNA.
The poly(A) tail is present at the 3’-ends of most eukaryotic mRNAs and their precursors.
Length: The tail is approximately $250$ nucleotides long.
Synthesis: It is added post-transcriptionally by an enzyme called poly(A) polymerase (PAP).
Source: Because it is added after transcription and is not encoded in the DNA template, the poly(A) tail is not a product of transcription.

The poly(A) tail provides three primary functions: 1. Enhances the lifetime of the mRNA. 2. Enhances the translatability of the mRNA. 3. Serves as a requirement for the efficient transport of mRNAs from the nucleus to the cytoplasm.
The relative importance of these three effects can vary between different biological systems.

Transcription Phase: Transcription of eukaryotic genes typically extends past the actual ‘polyadenylation site’.
Processing Phase: The transcripts are then cleaved and subsequently polyadenylated at the new 3’-end created by that cleavage.
Mammalian Polyadenylation Signal Motifs: A minimum efficient signal consists of three parts: 1. AAUAAA motif: Located approximately $20\,\text{nt}$ upstream of the polyadenylation site in the pre-mRNA. 2. GU-rich motif: Located $23$ or $24\,\text{bp}$ downstream of the AAUAAA motif. 3. U-rich motif: Follows immediately after the GU-rich motif.

Cleavage in mammals requires the coordination of several proteins: 1. CPSF (Cleavage and Polyadenylation Specificity Factor). 2. CstF (Cleavage Stimulation Factor). 3. CF I (Cleavage Factor I). 4. CF II (Cleavage Factor II). 5. Poly(A) polymerase. 6. RNA polymerase II.

Initiation of Polyadenylation: * Two proteins participate: CPSF (which binds to the AAUAAA motif) and Poly(A) polymerase. * The optimal signal for initiation on a cleaved substrate is the AAUAAA motif followed by at least $8\,\text{nt}$ . * Once the poly(A) tail reaches approximately $10\,\text{nt}$ in length, the process shifts; further polyadenylation becomes independent of the original AAUAAA signal and depends on the poly(A) tail itself.
Elongation of the Poly(A) Tail: * Requires a specificity factor called Poly(A)-Binding protein II (PAB II). * PAB II binds to the pre-initiated oligo(A) tail. * It assists poly(A) polymerase in elongating the tail to its final length of $250\,\text{nt}$ or more. * PAB II acts independently of the AAUAAA motif and depends only on the poly(A) sequence. * The activity of PAB II is further enhanced by CPSF.

Factor Assembly: Various factors assemble on the pre-mRNA, guided by the recognition of specific motifs.
Cleavage: RNA polymerase II stimulates the cleavage of the pre-mRNA.
Poly(A) Synthesis: Poly(A) polymerase initiates the synthesis of the tail.
Rapid Extension: PAB II enables the rapid extension of the oligo(A) to its full-length state.

All three major RNA processing events (capping, splicing, and polyadenylation) occur while transcription is still in progress.
Splicing: Splicing mechanisms begin while transcription is actively underway.
Capping Timing: Occurs when the nascent mRNA is about $30\,\text{nt}$ long, specifically when the 5’-end of the RNA first emerges from the RNA polymerase complex.
Polyadenylation Timing: Occurs when the still-growing mRNA is cut at the designated polyadenylation site.

The Carboxy-Terminal Domain (CTD) of the Rpb1 subunit of RNA polymerase II is a central hub for all three types of RNA processing.
The CTD serves as a platform or scaffold where capping, polyadenylating, and splicing enzymes bind directly.

Termination of transcription by RNA polymerase II involves two primary steps: 1. Co-transcriptional Cleavage (CoTC): The transcript is cleaved within the termination region downstream of the polyadenylation site. 2. Secondary Cleavage: Cleavage and polyadenylation occur at the poly(A) site, which provides the signal for the RNA polymerase to dissociate from the DNA template.