Exhaustive Notes on Eukaryotic RNA Processing and Post-Transcriptional Modification

Learning Objectives for RNA Processing

• Describe and place in order the three primary steps in eukaryotic post-transcriptional processing: $5'$ capping, intron splicing, and $3'$ polyadenylation.

• Explain the necessity of each step in RNA processing and the role each modification plays in ensuring mRNA can be translated.

• Describe the roles of specific RNA regions in the pre-mRNA and the enzymes involved in eukaryotic RNA processing.

• Explain how differences in transcription and splicing can lead to different protein products.

Why is RNA Processed?

• Reason 1: Export Signals

  • The $5'$ cap and $3'$ poly-A tail provide specific signals required to export mature mRNA transcripts from the nucleus into the cytoplasm.

• Reason 2: Stability and Protection

  • Both the $5'$ cap and the $3'$ poly-A tail protect the mRNA from degradation.

  • No $5'$ cap leads to degradation by RNases.

  • A short or missing poly-A tail results in rapid degradation.

• Reason 3: Translation Initiation

  • Processing adds structures essential for the translation of mRNA, facilitating ribosome binding and orientation.

• Reason 4: Removing Introns

  • Processing removes introns (non-coding sequences) and joins exons together to create a continuous Coding Sequence ($CDS$).

Overview of the Transcription-Processing Workflow

• Pre-mRNA Structure:

  • Contains the $5'$ untranslated region ($5' UTR$), the Start codon ($AUG$), the polypeptide-coding sequence (containing both exons and introns), the Stop codon ($UAA$), and the $3'$ untranslated region ($3' UTR$).

  • Features a polyadenylation signal sequence ($AAUAAA$) and a downstream U-rich region.

  • The region between the $AAUAAA$ and the U-rich region (approx. $15-30$ nucleotides) contains the cleavage site.

• Mature mRNA Structure:

  • Includes the $5'$ Cap, $5' UTR$, Start codon ($AUG$), the continuous coding region (introns removed), Stop codon ($UAA$), $3' UTR$, and the Poly-A tail ($20-200$ Adenine nucleotides).

The $5'$ Cap: Modification of the $5'$ End

• Definition: The addition of a $5'$ $7$-methylguanosine cap.

• Timing: Capping occurs co-transcriptionally after a short piece of RNA has been transcribed. The initial RNA molecule has a $5'$ triphosphate.

• Mechanism of Addition:

  • Enzyme Recruitment: Capping enzyme (guanylyl transferase) is recruited to the C-terminal domain ($CTD$) tail of RNA Polymerase II.

  • Bond Formation: An inverted guanosine is added via a unique $5'-5'$ triphosphate bond.

  • Methylation: RNA methyltransferase adds a methyl group ($CH_3$) to the added guanine at the $7$ position, and often to one or more subsequent bases.

• Functions of the $5'$ Cap:

  • Protects the transcript from degradation by RNases (increases stability).

  • Serves as a signal for nuclear export.

  • Facilitates intron splicing.

  • Promotes ribosome binding and proper orientation for translation.

Splicing: Modification of the Middle

• Concept: Removal of introns and ligation of exons. All nucleotides from the transcription start to the end are initially transcribed into pre-mRNA, making it longer than the mature mRNA.

• The Spliceosome:

  • A large, dynamic, and flexible complex consisting of proteins and small nuclear RNAs ($snRNAs$).

  • Composition: Humans have approximately $300$ different spliceosomal proteins and $100,000$ spliceosomes per cell.

  • $snRNPs$ (Small Nuclear Ribonucleoproteins): Pronounced "snurps," these are made of $snRNA$ ($100-200$ nucleotides long) and proteins. The RNA components contain sequences antiparallel and complementary to pre-mRNA consensus sequences or other $snRNAs$.

• Spliceosome Mechanics:

  • Recognition: The spliceosome recognizes consensus sequences/motifs at exon/intron boundaries, specifically the $5'$ splice site, the branch site (containing an Adenine), and the pyrimidine tract ($Cytosine$ or $Uracil$ rich).

  • Complementary Binding: For example, the $U_2$ RNA forms hydrogen bonds with the branch site.

• The Two-Step Splicing Reaction:

  • Reaction 1: Cleavage at the $5'$ splice site. The $5'$ end of the intron attaches to the Adenine at the branch point, forming a $2'-5'$ phosphodiester bond. This creates a loop structure called a lariat.

  • Reaction 2: Cleavage at the $3'$ splice site. The lariat (intron) is released and eventually degraded. Exon $1$ is joined to Exon $2$ through a new phosphodiester bond (ligation).

Alternative Splicing and Protein Diversity

• Definition: A process where a single gene or pre-mRNA can be spliced in different combinations of exons to produce multiple mRNA isoforms.

• Significance:

  • One gene can yield many different proteins (isoforms).

  • Isoforms may have similar functions but differ in specific amino acid blocks.

  • This is cell-type, tissue, or developmental stage-specific.

• Statistics: Mammalian genomes have approximately $20,000$ protein-coding genes but can produce over $100,000$ different proteins due to alternative splicing.

The Poly-A Tail: Modification of the $3'$ End

• Mechanism: Modification by cleavage and polyadenylation.

• Step 1: Recognition and Cleavage:

  • Polyadenylation Factors: A collective group of proteins recognize the $3'$ end motifs.

  • $CPSF$ (Cleavage and Polyadenylation Specific Factor): Binds to the $AAUAAA$ consensus sequence and catalyzes cleavage $15-30$ bases downstream.

  • $CStF$ (Cleavage Stimulation Factor): Binds to the U-rich region.

  • $CFI$ and $CFII$: Bind additional regions to promote cleavage at the cleavage site.

• Step 2: Polyadenylation:

  • $PAP$ (Polyadenylate Polymerase): Binds near $CPSF$ and adds adenine nucleotides to the $3'$ end. Note: $PAP$ must be bound for cleavage to occur, even though it performs the subsequent addition.

  • $PABs$ (Poly-A Binding Proteins): Help $PAP$ in the nucleus and remain bound to the tail to promote export, stability, and translation.

  • Tail Length: Typically $20-200$ Adenine nucleotides ($As$).

• Step 3: Termination of Transcription:

  • Cleaving the RNA to add the poly-A tail releases the mRNA. The remaining RNA piece bound to the RNA polymerase triggers the recruitment of Torpedo RNase, which degrades the bound RNA and terminates transcription.

RNA Editing

• Definition: Editing of the mRNA coding sequence at specific positions after transcription. This can include inserting Uracils ($Us$), changing Cytosine ($C$) to Uracil ($U$), or changing Adenine ($A$) to Inosine ($I$).

• Prevalence: Rare in many animals (including humans) but common in plants and cephalopods (e.g., octopuses and squid), allowing them to recode RNA to adapt to their environment.

Summary of Key Players

• Capping Enzyme: Guanylyl transferase that adds guanosine to the $5'$ end.

• RNA Methyltransferase: Methylates the guanosine and downstream nucleotides.

• Spliceosome/$snRNPs$: Catalyze intron removal and exon ligation.

• Polyadenylation Factors ($CPSF$, $CStF$, $CFI$, $CFII$): Recognize signal sequences and cleave the $3'$ end.

• $PAP$ (Polyadenylate Polymerase): Adds the adenine tail.

• $PAB$ (Poly-A Binding Protein): Protects the tail and aids in export and translation.

Questions & Discussion

• Question: How do bacterial and eukaryotic gene transcripts differ in terms of modification?

  • Response: Bacterial mRNAs are often polycistronic (multiple genes per transcript) and are not modified (no cap, splicing, or poly-A tail). Eukaryotic transcripts are monocistronic (one gene per mRNA) and undergo extensive modification ($5'$ cap, splicing, $3'$ poly-A tail) before translation.

• Question: How does a single base-pair substitution at the end of an intron cause a mutation in a protein like $\beta$-globin?

  • Response: If the mutation occurs at a recognized splice site boundary, the spliceosome may fail to recognize the site. This can lead to the retention of the intron or the exclusion of an exon, altering the final protein sequence.

• Question: What other non-exon DNA changes could produce disease?

  • Response:

    1. Promoter: Decrease or stop transcription.

    2. Terminator: Elongate the transcript or decrease functional protein.

    3. Enhancer: Decrease transcription levels.

    4. Silencer: Increase transcription levels unnecessarily.