RNA Processing, Splicing, and Translation: Key Concepts in Eukaryotic Gene Expression

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RNA Processing and Turnover

Primary Transcript and RNA Processing

Newly synthesized RNA molecules, known as primary transcripts, must undergo chemical modifications before functioning in the cell. This process, called RNA processing, generates a mature RNA product and may include association with specific proteins or export from the nucleus in eukaryotes.

Primary transcript: The initial RNA product from transcription.
RNA processing: Chemical modifications such as capping, polyadenylation, and splicing.
Mature RNA: The functional RNA after processing.

Messenger RNA Processing in Eukaryotes

Capping, Poly(A) Addition, and Intron Removal

Eukaryotic mRNA undergoes extensive processing, including the addition of a 5' cap, a 3' poly(A) tail, and removal of introns. In contrast, most bacterial RNA is synthesized in a form ready for translation, with no need for processing due to the absence of a nuclear membrane.

5' Cap: Modified guanosine added to the 5' end, methylated at position 7.
Poly(A) Tail: Long stretch of adenines (50–250 nucleotides) added to the 3' end by poly(A) polymerase.
Introns: Non-coding sequences removed from pre-mRNA.

Transcription and Translation in Eukaryotes

Export and Processing of Eukaryotic Transcripts

Eukaryotic transcripts must be exported from the nucleus for translation. Substantial processing occurs in the nucleus, and primary transcripts (heterogeneous nuclear RNA, hnRNA) are often very long (2000–20,000 nucleotides).

hnRNA: Mixture of mRNA molecules and their precursors (pre-mRNA).
Pre-mRNA: Processed by removal of introns and addition of 5' caps and 3' tails.

5' Caps and 3' Poly(A) Tails

Structure and Function

The 5' cap and 3' poly(A) tail are essential modifications for eukaryotic mRNA stability, export, and translation.

5' Cap: Guanosine methylated at position 7, bound by a 5'–5' linkage.
Poly(A) Tail: Added downstream of the AAUAAA signal by poly(A) polymerase; GU- or U-rich elements are located downstream.

Roles of the 5' Cap:

Added soon after transcription initiation.
Protects mRNA from nucleases.
Positions mRNA on the ribosome for translation initiation.

Function of the Poly(A) Tail:

Protects mRNA from nuclease attack; tail length influences stability.
Required for export to the cytoplasm.
May help ribosomes recognize and bind mRNAs.

Exons and Introns

Definitions and Variability

Exons are sequences that appear in the final mRNA, while introns are non-coding regions present in most protein-coding genes of multicellular eukaryotes. The size and number of introns vary considerably.

Exons: Coding sequences retained in mature mRNA.
Introns: Non-coding sequences removed during processing.

Spliceosomes and RNA Splicing

Mechanism and Components

RNA splicing removes introns and joins exons. Splicing errors can lead to inherited diseases. Splice sites are determined by conserved sequences at intron-exon boundaries.

5' splice site: Typically starts with GU.
3' splice site: Typically ends with AG.
Branch point: Contains an A residue near the 3' end of the intron.

Spliceosome: Large complex of five types of RNA and over 200 proteins, assembled from snRNPs (small nuclear ribonucleoprotein complexes).

Spliceosome Assembly

U1 snRNP binds the 5' splice site.
U2 snRNP binds the branch-point sequence.
U4/U6 and U5 snRNPs bring intron ends together, forming a mature spliceosome.
Cleavage at the 5' splice site joins it to the branch-point A, forming a lariat structure.
Cleavage at the 3' splice site joins exons; exon junction complex (EJC) is deposited at exon-exon boundaries.

Classes of Introns

99% use the U2 spliceosome (GU-AG introns).
Second class (AU-AC introns) excised by a different spliceosome using U12 snRNP.

Alternative Splicing

Mechanisms and Regulation

Alternative splicing allows a single gene to produce multiple protein products by varying the combination of exons included in the mature mRNA.

Regulatory proteins and snoRNAs bind to splicing enhancer or silencer sequences.
Most snoRNAs direct modifications to tRNAs and rRNAs.

Nuclear Export of Mature mRNAs

Export Mechanism

Mature mRNA must exit the nucleus via the nuclear pore complex (NPC) for translation. Only fully processed transcripts are exported, providing an additional regulatory layer.

Nuclear RNA export factor 1 (NXF1): Interacts with cap-binding complex (CBC) and exon junction complex (EJC) to facilitate export.
Export involves many additional factors, including eIF4E.

Translation: The Genetic Code and Machinery

The Genetic Code

The genetic code is a triplet code, with three nucleotide bases specifying each amino acid. There are 64 possible codons, 61 of which code for amino acids and 3 for stop signals.

Degenerate code: Multiple codons can specify the same amino acid.
Nonoverlapping: Reading frame advances three nucleotides at a time.
Unambiguous: Each codon specifies only one amino acid.
Nearly universal: Used by almost all organisms, with few exceptions (e.g., selenocysteine, mitochondrial codes).

Codon Table

Codon	Amino Acid	Function
AUG	Methionine	Start codon
UAA, UAG, UGA	None	Stop codons
Other codons	Various	Specify amino acids

Translation Machinery

Ribosomes: Carry out polypeptide synthesis; composed of large and small subunits (rRNA and proteins).
tRNA: Aligns amino acids in the correct order; each tRNA is linked to its amino acid by an ester bond.
Aminoacyl-tRNA synthetases: Attach amino acids to their appropriate tRNA molecules using ATP hydrolysis.
mRNA: Encodes the amino acid sequence information.
Protein factors: Facilitate steps of translation.

Ribosome Sites

Site	Function
A (aminoacyl) site	Binds tRNA with attached amino acid
P (peptidyl) site	Holds tRNA carrying the growing peptide
E (exit) site	tRNAs leave after discharging their amino acid

Transfer RNA (tRNA) Structure and Function

Adaptor molecule: Binds both a specific amino acid and the mRNA codon specifying that amino acid.
Aminoacyl tRNA: tRNA attached to an amino acid (charged tRNA).
Anticodon: Sequence in tRNA complementary to the mRNA codon, allowing recognition via base pairing.

Aminoacyl-tRNA Synthetases

20 different synthetases, one for each amino acid.
Catalyze attachment of amino acids to tRNA using ATP:

Proofreading ensures correct amino acid is added.

Messenger RNA (mRNA) and Translation Initiation

mRNA sequence of codons directs amino acid order.
Untranslated regions (UTRs) at 5' and 3' ends are essential for mRNA function.
Start codon (AUG) initiates translation; stop codons (UAA, UAG, UGA) terminate it.

Translation Mechanism

Translation begins at the N-terminus and proceeds to the C-terminus.
mRNA is read in the 5' → 3' direction.

Bacterial Initiation

Three initiation factors (IF1, IF2, IF3) bind the small ribosomal subunit (30S).
mRNA binds via the Shine-Dalgarno sequence, aligning the start codon at the P site.
Initiator tRNA carries N-formylmethionine (fMet).
Large subunit (50S) joins to form the 70S initiation complex.

Eukaryotic Initiation

Start codon specifies methionine (not formylated).
Initiation factors are called eIFs; about a dozen are involved.
Initiator tRNA distinct from elongation tRNAMet.
Some mRNAs use internal ribosome entry sequences (IRES) for ribosome recruitment.

Activity: DNA Repair Mechanisms

List 4 Mechanisms of DNA Repair and Proteins Involved

Direct Reversal: Enzymes such as photolyase repair UV-induced thymine dimers.
Base Excision Repair (BER): DNA glycosylases, AP endonuclease, DNA polymerase, DNA ligase.
Nucleotide Excision Repair (NER): UvrABC endonuclease complex, DNA polymerase, DNA ligase.
Mismatch Repair (MMR): MutS, MutL, MutH proteins in prokaryotes; MSH, MLH, PMS proteins in eukaryotes.

Additional info: These repair mechanisms are essential for maintaining genome integrity and preventing mutations that could lead to disease.