RNA Modification

Colinearity of Gene Expression and Non-Colinearity in Eukaryotes

• Bacterial Colinearity:

  • Bacterial gene expression follows direct colinearity, where the linear sequence of DNA nucleotides directly corresponds to the codon sequence in mRNA, which in turn corresponds directly to the amino acid sequence of the synthesized polypeptide.
  • DNA Coding Strand Example: $5'-\text{ATG GAA TTT}-3'$
  • mRNA Sequence Example: $5'-\text{AUG GAA UUU}-3'$
  • Polypeptide Sequence Example: $\text{Met-Glu-Phe}$
  • RNA transcription replaces Thymine ($\text{T}$) present in DNA with Uracil ($\text{U}$).

• Eukaryotic Non-Colinearity:

  • Discoveries in the late 1970s demonstrated that eukaryotic genomic DNA sequences are not continuously colinear with their corresponding mature mRNA transcripts.
  • Eukaryotic genes are composed of interrupted sequence architecture:
  • Exons: Coding sequences retained in the final mature RNA product.
  • Introns: Intervening non-coding sequences located between exons that do not code for protein sequences.

• RNA Splicing Mechanism:

  • Transcription of eukaryotic genomic DNA produces a primary precursor transcript known as pre-mRNA (or primary transcript), which contains both exon and intron sequences in sequence ($\text{Exon 1}-\text{Intron}-\text{Exon 2}-\text{Intron}-\text{Exon 3}$).
  • RNA Splicing: A fundamental post-transcriptional process wherein introns are excised (removed) and exons are covalently joined together.
  • The resulting mature mRNA contains only connected exons ($\text{Exon 1}-\text{Exon 2}-\text{Exon 3}$) and is no longer continuously colinear with the genomic DNA sequence.
  • RNA splicing is a widespread genetic phenomenon in eukaryotes and occurs occasionally in bacteria.

Monocistronic versus Polycistronic mRNA Structural Organization

• Eukaryotic Monocistronic mRNA:
  • Eukaryotic mRNAs are predominantly monocistronic, meaning each mature mRNA transcript contains a single major protein-coding open reading frame (ORF) and directs the synthesis of one protein species.
  • Structural components of eukaryotic monocistronic mRNA:
  • $5'$ cap structure ($\text{m}^7\text{G}$ attached via a $5'-5'$ triphosphate linkage).
  • $5'$ Untranslated Region ($5'$ UTR).
  • Translation Start Codon ($\text{AUG}$).
  • Protein-coding region (exons).
  • Translation Stop Codon ($\text{UAA}$, $\text{UAG}$, or $\text{UGA}$).
  • $3'$ Untranslated Region ($3'$ UTR).
  • $3'$ Poly(A) tail (a stretch of adenine residues).

• Prokaryotic Polycistronic mRNA:
  • Prokaryotic genes involved in coordinated functional pathways are frequently grouped into operons transcribed as a single transcriptional unit.
  • Prokaryotic mRNAs are polycistronic, encoding multiple distinct protein-coding regions within a single transcript.
  • Structural components of prokaryotic polycistronic mRNA:
  • Multiple open reading frames (e.g., Gene A, Gene B, Gene C), each bounded by its own start codon ($\text{AUG}$) and stop codon ($\text{UAA}$ or $\text{UAG}$).
  • Intercistronic regions: Non-coding spacer sequences located between individual coding regions.
  • Translation yields multiple separate proteins (e.g., Protein A, Protein B, Protein C) simultaneously from one mRNA.

Processing and Maturation of Mammalian Ribosomal RNA

• Pre-rRNA Synthesis and Subunit Components:
  • Ribosomal DNA (rDNA) exists in repeated units transcribed in the nucleolus by RNA Polymerase I into a large primary transcript termed **45\text{S}$ pre-rRNA**.\n * A single 45\text{S} pre-rRNA primary transcript undergoes extensive processing to yield three mature ribosomal RNAs:\n * 18\text{S} rRNA\n * 5.8\text{S} rRNA\n * 28\text{S} rRNA\n * **5\text{S}$rRNA** is not part of the$45\text{S} pre-rRNA transcript; it is transcribed separately outside the nucleolar organizer region by **RNA Polymerase III**.\n\n* **Cleavage, Trimming, and Nucleoside Modifications**:\n * **Spacer Removal**: Non-coding spacer sequences are cleaved and degraded:\n * External Transcribed Spacers (5'$ETS and$3' ETS).\n * Internal Transcribed Spacers (ITS1 and ITS2).\n * **Covalent Nucleoside Modifications**:\n * Nucleosides undergo extensive post-transcriptional chemical modifications, primarily **2'-O\text{-methylation}** and **pseudouridylation**.\n * These chemical modifications are guided site-specifically by **small nucleolar RNAs (snoRNAs)**.\n\n* **Ribosome Subunit Assembly and Structural Composition**:\n * Processing of pre-rRNA and early assembly steps take place in the nucleolus.\n * **40\text{S} Small Ribosomal Subunit**:\n * Composed of mature 18\text{S}$rRNA plus$33 ribosomal proteins.\n * **60\text{S} Large Ribosomal Subunit**:\n * Composed of mature 5.8\text{S}$rRNA,$28\text{S}$rRNA, and$5\text{S}$rRNA plus approximately$46 ribosomal proteins.\n * **80\text{S} Mature Ribosome**:\n * Assembly of one 40\text{S}$subunit and one$60\text{S}$subunit forms the fully functional eukaryotic$80\text{S} ribosome.\n\n\n\n# Transfer RNA Processing and Base Modification\n\n* **Pre-tRNA Cleavage and Intron Removal**:\n * Transfer RNAs are initially synthesized as precursor tRNAs (pre-tRNAs) containing extra non-coding sequences at both ends and, in some cases, introns.\n * **Intron Excision Pathway**:\n * The endonuclease complex **TSEN/CLP1** cleaves pre-tRNA at specified exon-intron boundaries to liberate the intron.\n * Cleavage generates a 5'\text{-exon}$fragment with a$3'\text{-phosphate}$terminus and a$3'\text{-exon}$fragment with a$5'\text{-hydroxyl}$($\text{OH}) terminus.\n * **tRNA Ligase** covalently joins the 5'\text{-exon}$and$3'\text{-exon} together to form a continuous mature tRNA structure.\n\n* **End Trimming and Ribozyme Catalysis**:\n * **5'$End Processing**: **RNaseP**, an endonuclease complex composed of RNA and protein subunits, cleaves the pre-tRNA to establish the mature$5' end.\n * The RNA subunit of RNaseP contains intrinsic catalytic activity, defining RNaseP as a **ribozyme**.\n * **3'$End Processing**: **RNaseD**, an exonuclease, trims$9$nucleotides from the$3' terminal region.\n * **3'\text{-CCA}$Terminal Sequence**: The invariant sequence$3'\text{-CCA} serves as the amino acid attachment site, where an amino acid is covalently charged onto the terminal adenosine residue.\n\n\n\n* **Covalently Modified Bases in tRNA**:\n * Mature tRNAs contain extensive post-transcriptionally modified bases crucial for proper secondary and tertiary folding, structural stability, codon-anticodon recognition, and translational fidelity:\n * Methylguanosine (\text{m}^7\text{G}$or$\text{m}\text{G})\n * 2-Isopentenyladenosine (IP)\n * Pseudouridine (P)\n * 4-Thiouridine (T)\n\n# 5' Capping of Eukaryotic mRNA\n\n* **Timing and Structural Characteristics**:\n * Capping involves the covalent attachment of a **7-methylguanosine** (\text{m}^7\text{G}$) to the$5' terminal nucleotide of eukaryotic pre-mRNA.\n * Occurs **co-transcriptionally** as the transcript is being synthesized by RNA Polymerase II, typically when the nascent RNA transcript is only 20 \text{ to } 25 nucleotides long.\n * The cap structure is attached via an unusual **5'-5' triphosphate linkage**.\n\n* **Enzymatic Reactions of Capping**:\n 1. **RNA 5′-triphosphatase**: Cleaves and removes the \gamma\text{-phosphate}$from the$5' terminal triphosphate group of the initial pre-mRNA transcript.\n 2. **Guanylyltransferase**: Hydrolyzes GTP into GMP by removing two phosphate groups (pyrophosphate release) and attaches GMP to the remaining 5' diphosphate end.\n 3. **Methyltransferase**: Transfers a methyl group (\text{CH}_3$) from S-adenosylmethionine to the N7 position of the terminal guanine base, generating the mature$7\text{-methylguanosine} cap structure.\n\n* **Biological Consequences and Functions of the 5' Cap**:\n * **Protection from Degradation**: Shields the mRNA 5'$end from cleavage by$5' \rightarrow 3' exonucleases.\n * **Promotion of Pre-mRNA Processing**: Facilitates efficient pre-mRNA splicing, particularly for the excision of the first intron, and supports 3' end processing.\n * **Nuclear Export**: Recognized by cap-binding complexes that promote the transport of mature mRNA through nuclear pore complexes into the cytoplasm.\n * **Translation Initiation**: Cytoplasmic cap-binding proteins recognize the 5'$cap and recruit translation initiation factors along with the$40\text{S} ribosomal subunit to begin protein synthesis.\n\n# 3' Polyadenylation of Eukaryotic mRNA\n\n* **Structural Characteristics**:\n * Most mature eukaryotic mRNAs feature a continuous tail of adenine nucleotides at their 3' end, termed the **poly(A) tail**.\n * The poly(A) tail is **not encoded in the genomic DNA sequence**; it is added post-transcriptionally through enzymatic activity following transcription completion.\n\n* **Two-Step Mechanism of Polyadenylation**:\n 1. **Endonucleolytic Cleavage**:\n * The consensus **polyadenylation signal** sequence (5'-\text{AAUAAA}-3'$) is located upstream of the cleavage site in the$3' UTR.\n * Endonucleolytic cleavage occurs downstream of the 5'-\text{AAUAAA}-3' signal sequence, typically at a **CA** dinucleotide cleavage site.\n * The downstream RNA fragment generated past the cleavage site is degraded.\n 2. **Poly(A) Addition**:\n * **Poly(A) Polymerase (PAP)** catalyzes the addition of approximately 100 \text{ to } 250$adenine residues ($\text{A}$residues) to the newly created$3'-\text{OH} terminus.\n * PAP uses ATP as a substrate and operates in a template-independent manner (without a DNA template).\n\n\n\n* **Functional Role of the Poly(A) Tail**:\n * Dramatically increases mRNA transcript stability by retarding enzymatic degradation.\n * Enhances translation efficiency by promoting mRNA interaction with translational machinery in the cytoplasm.\n\n# RNA Splicing Mechanisms and Self-Splicing Ribozymes\n\n* **Overview of Splicing Pathways**:\n * Three primary categories of splicing mechanisms exist:\n 1. **Group I intron splicing**\n 2. **Group II intron splicing**\n 3. **Spliceosome-mediated splicing**\n * All three pathways accomplish two fundamental catalytic steps:\n * Precision excision/removal of the intron RNA sequence.\n * Covalent linkage of adjacent exon RNA segments via a phosphodiester bond.\n\n* **Self-Splicing Principles (Group I and Group II Introns)**:\n * Splicing in Group I and Group II introns is termed **self-splicing** because it requires no protein enzymes or spliceosomal complexes for catalytic turnover.\n * The intron RNA itself adopts a specific tertiary fold that functions as a catalytic **ribozyme**.\n * Prevalent in primary ribosomal RNA (rRNA) and transfer RNA (tRNA) transcripts in organellar and bacterial systems.\n * Group I and Group II introns differ in their chemical mechanism of intron excision and exon joining.\n\n* **Group I Intron Self-Splicing Reaction Mechanism**:\n * Self-splicing of Group I introns proceeds through two successive transesterification steps initiated by an exogenous guanosine nucleoside cofactor (free G, GMP, GDP, or GTP):\n 1. **First Transesterification Reaction**:\n * An external free guanosine nucleoside binds to a specific guanosine-binding pocket located within the Group I intron fold.\n * The 3'-\text{OH}$group of the bound guanosine attacks the phosphodiester bond at the$5' splice junction (between Exon 1 and the intron).\n * The cleavage breaks the bond between Exon 1 and the intron, covalently attaching the external guanosine to the 5' end of the excised intron segment.\n 2. **Second Transesterification Reaction**:\n * The exposed free 3'-\text{OH}$terminus of Exon 1 attacks the phosphodiester bond at the$3' splice junction (between the intron and Exon 2).\n * Exon 1 and Exon 2 become covalently linked through a standard phosphodiester bond.\n 3. **Products**:\n * Covalently joined mature exon RNA (\text{Exon 1}-\text{Exon 2}).\n * A linear intron RNA bearing the exogenous guanosine residue at its 5'$$ terminus.