cbg 14- RNA Processing: Capping, Tailing, and Splicing

Overview of Post-Transcriptional Processing

• Universal Processing: tRNA and rRNA are heavily post-transcriptionally processed in all living organisms.

• Processing of tRNA:     * Methylation: Chemical modification of bases.     * Pseudouridylation: The conversion of uridine to pseudouridine.

• Processing of rRNA:     * Cleavage: Cutting the primary transcript into functional units.     * Methylation: Chemical modification for structural stability.     * Eukaryotic Ribosome Synthesis: Occurs within the nucleolus.     * Genomic Organization: In Homo sapiens, there are multiple tandem repeats of rDNA to ensure sufficient production.     * Efficiency: There is no translational amplification for rRNA; the RNA itself is the final product.     * Visualizing Transcription: "Christmas trees" are observed in electron micrographs where multiple tandem repeats (e.g., 2 tandem repeats) are being transcribed from left to right by approximately 100 RNAP-III (RNA Polymerase III) molecules each.

Comparative mRNA Anatomy: Prokaryotes vs. Eukaryotes

• Prokaryotic mRNA (Bacteria):     * Polycistronic: Often contains several coding regions (cistrons) within a single mRNA molecule.     * Processing: No significant mRNA processing occurs; transcription and translation can happen concurrently.     * Key Features:         * Shine-Dalgarno Sequence (SDS): Located in the $5'\text{-UTR}$, it serves as the ribosome binding site for each coding region.         * Structure: 5'\text{-SDS-START-CODE#1-STOP---SDS-START-CODE#2-STOP-}3'

• Eukaryotic mRNA:     * Monocistronic: Only one coding region per mRNA molecule.     * Significant Processing: Extensive modifications occur in the nucleus before export.     * Key Features:         * $5'$ Cap: A methylated guanosine ($m^7G$) cap.         * $5'\text{-UTR}$ and $3'\text{-UTR}$: Untranslated regions flanking the coding sequence.         * Start Codons: Primarily $AUG$, occasionally $GUG$.         * Stop Codons: $UAA$, $UGA$, and $UAG$.         * $3'$ Poly-A Tail: A string of approximately 200 adenosine residues ($A_{200}$).

mRNA Processing in Eukaryotes: Capping and Tailing

• $5'$ Capping ($m^7G$ Cap):     * Function: Provides protection from degradation within the nucleus and serves as a ribosome binding site for the initiation of translation.

• $3'$ Polyadenylation (Poly-A Tail):     * Origin: Added post-transcriptionally; it is not directly encoded in the DNA template.     * Functions:         * Stability: Slows down degradation in the cytoplasm (the tail is gradually removed by deadenylating nuclease).         * Nuclear Export: Facilitates transport of the mRNA through nuclear pores.         * Targeting: Assists in the delivery of mRNA to specific locations within the cell.

• Mechanism of Polyadenylation:     1. Cleavage: Factors like CPSF (Cleavage and Polyadenylation Specificity Factor) and CStF (Cleavage Stimulation Factor) recognize the sequence $AAUAAA$, followed by a $CA$ cleavage site and a downstream $GUGGUUGGUU$ (GU-rich) region.     2. Termination: Cleavage at the $CA$ site terminates transcription.     3. Tail Addition: Poly-A Polymerase (PAP) adds the adenosine tail in the nucleus.     4. Ribonucleoprotein Formation: Poly-A Binding Proteins (PABP) coat the tail.     5. Cytoplasmic Fate: In the cytoplasm, deadenylating nuclease removes A residues. When the tail is short enough, the mRNA is destroyed by the exosome.

mRNA Stability and Cytoplasmic Targeting

• Determining Half-Life ($t_{1/2}$):     * The length of the poly-A tail is a primary determinant of an mRNA molecule's stability.     * Tug-of-War: Cytoplasmic Poly-A Polymerase (PAP) attempts to extend the tail while Deadenylating Nuclease (DAN) attempts to shorten it.

• Stability Examples:     * $\beta\text{-globin}$ (Red Blood Cells): Highly stable, with a $t_{1/2} \approx 10\,\text{hr}$.     * Typical mRNA: Moderate stability.     * Histone mRNA: Lacks a poly-A tail, possessing a stem-loop structure instead.         * S phase: Stable during DNA synthesis.         * $G_2$ phase: Stem-loop binding protein is degraded, leading to rapid degradation of histone mRNA.

• Targeting via the $3'\text{-UTR}$:     * Specific sequences in the $3'\text{-UTR}$ target mRNA to distinct cytoplasmic regions.     * Example: Vasopressin mRNA is directed to dendrites in the hypothalamus. This involves PABP binding to cytoskeletal motors which transport the mRNA from the cell body along the axon or to dendrites.

Splicing: Mechanisms and Components

• Definitions:     * Introns: "Junk" DNA (non-coding sequences) removed from pre-mRNA.     * Exons: Coding sequences ligated together to form mature mRNA.

• The Splicing Process:     * Pre-mRNA contains Intron ($I$) and Exon ($E$) sequences (e.g., $E1-I1-E2-I2…$).     * Consensus Sequences: Splicing occurs at the Donor site ($GU$), the Branch point ($A$) within a branch/bridge sequence (e.g., $UAAC$), and the Acceptor site ($AG$).     * Chemical Reaction: The spliceosome activates the adenosine-OH (hydroxyl group) in the branch point to perform a nucleophilic attack on the phosphodiester bond at the donor site.     * Product: The intron is released as a "lariat" structure and discarded, while exons are spliced together.

• The Spliceosome:     * A massive complex composed of snRNP (Small Nuclear Ribonucleoprotein) "U particles."     * U1: Binds the $5'$ donor site.     * U2: Binds the bridge/branch point sequence.     * U4/U6: Re-checks the donor site.     * U5: Binds the $3'$ acceptor site.

• Evolutionary Theory: The spliceosome likely evolved from self-splicing bacterial introns, possibly introduced to eukaryotes as parasites by the protomitochondrion.

The mRNA Factory: RNAP-II Coordination

• C-terminal Domain (CTD): The CTD of RNA Polymerase II (RNAP-II) acts as a "staging post" or scaffold for processing factors.

• Temporal Process:     * Capping: The $5'$ cap is added by the Cap-Binding Complex (CBC) as soon as the $5'$ end exits RNAP-II.     * Splicing: Spliceosomes (U particles) associate with the CTD and splice exons as they "spool out" of the polymerase.     * Tailing: Tailing factors (like CPSF) are positioned on the CTD to polyadenylate the mRNA and signal the termination of transcription.

Alternative Splicing and Diversity

• Definition: The use of different splice sites to produce multiple distinct proteins from a single gene sequence.

• Types of Alternative Splicing:     * Standard Splice: Normal exon-to-exon joining.     * Exon Skipping: An exon is omitted from the final transcript.     * Intron Retention: An intron is left in the transcript and translated.

• Genomic Comparison:     * $Escherichia\ coli$: Average gene size is $1,000\,\text{bp}$; approximately $4,377$ genes produce roughly $4,300$ distinct polypeptides.     * $Homo\ sapiens$: Average gene size is $30,000\,\text{bp}$; approximately $25,000$ genes produce an estimated $30,000$ to $90,000$ distinct polypeptides due to alternative splicing.

• Functional Example: Immunoglobulins:     * A single immunoglobulin gene can produce either a membrane-bound B-cell receptor or a soluble antibody through the interplay of splicing and tailing.     * B-cell receptor: A T-cell stimulus induces a Cell-specific Transcription Factor. Splicing retains a hydrophobic tail and removes an early stop codon, resulting in a membrane-bound protein.     * Soluble antibody: In the absence of specific conditions or when using a weak CPSF binding site, a truncated transcript is produced. It cannot splice out the early stop codon and lacks the hydrophobic tail, resulting in a soluble antibody.

Questions and Discussion

• Q: Which of the following species of RNA are transcriptionally modified?     * A: mRNA, tRNA, and rRNA (All of them).

• Q: What’s a function of the $3'\text{-UTR}$ on mRNA?     * A: A. Tag mRNA for transport (Also involved in stability, but not directly for splicing).

• Q: What’s the function of deadenylating nuclease in mRNA processing?     * A: B. To remove adenosine tails (leading eventually to mRNA degradation).

• Q: Where does splicing (mostly) occur?     * A: In the nucleus (It is a nuclear process before export to the cytoplasm).

Summary of RNA Processing

• DNA replication initiates at origins of replication.

• In eukaryotes, mRNA modifications include the methyl-guanosine cap, the poly-A tail, and the removal of introns via spliceosomes.

• Alternative splicing allows a single gene to encode variant proteins.

• mRNA molecules are targeted by localized signals in the $3'\text{-UTR}$, and their longevity in the cytoplasm is determined by the length and presence of the poly-A tail.

concise

tRNA and rRNA undergo universal post-transcriptional processing in all organisms, which includes modifications such as methylation and pseudouridylation for tRNA, and cleavage and methylation for rRNA. Eukaryotic ribosome synthesis occurs in the nucleolus, with genomic organization ensuring sufficient rRNA production. Prokaryotic mRNA is polycistronic with concurrent transcription and translation, while eukaryotic mRNA is monocistronic and undergoes significant nuclear processing, including 5' capping and 3' polyadenylation for stability and nuclear export. The length of the poly-A tail influences mRNA stability, with specific cytoplasmic targeting through 3' untranslated regions (UTRs). Splicing occurs via spliceosomes to remove introns and join exons, generating mature mRNA. Alternative splicing allows diversification of proteins from single genes, illustrated by immunoglobulins producing different protein forms. Overall, these processes ensure the proper maturation, stability, and functioning of mRNA within the cell.