Splicing Mechanism of Pre-mRNA

RNA and Gene Expression

• RNA is a central molecule in gene expression.
• Life may have evolved from an RNA world.
• RNA molecules are negatively charged.
• RNA tends to repel one another in solution.
• Stable RNA secondary structures are formed in solution.
• RNA helix formation is stimulated by positive ions (e.g., Mg2+).

Types of RNA

• RNA is categorized as non-coding (ncRNA) or coding.
• Non-coding RNA includes housekeeping, regulatory, long, and small non-coding RNAs.
• Coding RNA includes mRNA, circular RNA (circRNA), microRNA (miRNA).
• Small ncRNAs include small nuclear RNA (snRNA), small nucleolar RNA (snoRNA).
• siRNAs (small interfering RNAs) and piRNAs (PIWI-interacting RNAs) are also present.

RNA Structures

• RNA has primary, secondary, and tertiary structures.
• Hydrogen bonding between complementary bases creates secondary structure.
• Secondary structures are formed hierarchically.
• Tertiary structures are built upon secondary ones.
• Elements of RNA structure include duplex, stem loops, bulges, internal loops.

RNA Processing: Intron Removal-Splicing

• Eukaryotic genes are interrupted (interrupted genes, interrupted genes).
• Exons are spliced together into a pre-mRNA by the spliceosome.
• Introns are not junk; they have regulatory elements and genes.
• Introns are longer than the final cytoplasmic mRNA in most eukaryotic genes.
• Intron removal plays a crucial role (removal, role) in RNA processing.
• RNA, polymerase transcription, gene expression, and timing are involved.

Alternative Splicing

• Alternative splicing occurs in most multicellular eukaryotes.
• Different splicing modes like intron retention or exon skipping contribute to protein diversity.
• Alternative splicing affects gene expression.
• Alternative splicing affects structural and functional diversity in gene products.

Types of RNA Splicing

• Nuclear RNA encoding proteins (cis-splicing) which requires conserved sequences.
• Trans-splicing of nuclear RNA, self-splicing, and type/group or type group II are also considered.

Introns in Eukaryotic Gene Expression

• Introns enhance eukaryotic gene expression by containing cis-acting elements that promote transcription.
• Introns can contain protein-coding or non-coding RNA genes, such as snoRNAs, miRNAs.
• Introns contribute to gene expression timing.
• Introns have an important role in the evolution of new proteins.

Capping of mRNA

• About 30 nt downstream of the initiation site in the transcribed mRNA, the RNA polymerase is paused.
• Pol II is paused, awaiting the recruitment of capping enzymes to complete its function after transcription initiation.
• A 5' terminal G-base is added to the mRNA by guanylyl transferase and linked to the terminal phosphate of the mRNA.
• Important for mRNA stability, splicing and export, and translation.

The Spliceosome

• A large, macromolecular complex, consisting of several non-coding RNAs and dozens of auxiliary proteins (snRNPs), is required for splicing.
• U1 to U6 are snRNAs, high in uridine residues.
• Components like Sm proteins and splicing factors are key players.
• The complex assembles and disassembles on each intron during RNA expression.

The Three Variant Spliceosomes

• The major spliceosome recognizes introns with a 5'-GT...AG-3' sequence.
• It has the essential snRNPs: U1, U2, U4/U6, and U5.
• The minor spliceosome recognizes introns with a 5'-AT...AC-3' sequence.
• Trans-spliceosome: facilitates splicing of separate pre-mRNA molecules transcribed from different genes.

Rules for Intron and Exon Definition

• Introns <200-500 nt: intron definition.
• Introns >500 nt: exon definition (critical for alternative splicing and various diseases).
• Exon definition is important for size, flanked by 5′ and 3′ splice sites, and gene expression control.

Splicing: A Two-Step Chemical Reaction

• A lariat is formed by intron cleavage and joining to a branch site adenosine's 2′ hydroxyl.
• The 3' end of the intron joins the 5' end of the next exon in a transesterification reaction.
• Splicing generates an intron lariat that's released and the exons are connected.

Splice Sites

• High conservation of nucleotides is observed within introns and at splice sites.
• Different splicing patterns are categorized based on the machinery involved.
• These patterns influence protein production.

Intron Removal Overview: Spliceosome Assembly

• ATP consumption helps assemble the spliceosome for lariat formation and intron release.
• The spliceosome undergoes multiple steps to remove introns: from precursor to active, then inactive form.
• Important reaction for releasing introns.

Spliceosome Assembly and Commitment

• Complementary sequences between snRNAs and pre-mRNA splice sites guide spliceosome assembly.
• The spliceosome assembles on the pre-mRNA, proceeds through various complexes, and ultimately catalyzes splicing reactions.
• Splicing proceeds through various molecular complex modifications to perform catalysis.

The Precursor Messenger RNA (Pre-mRNA) Splicing Cycle

• Splicing cycle involves multiple steps including pre-mRNA processing, intron excision, and final mRNA maturation.
• The cycle involves various regulatory proteins to ensure accurate mRNA sequence.

Spliceosome Cycle Steps 1-3

• Initiation of splicing involves association of pre-mRNA and hnRNPs (splicing regulators) to snRNPs (snRNA and proteins).
• Formation of the E-complex, based on the binding of U1 snRNP to the 5' splice site.
• Formation of the A-complex, which involves replacing splicing factors with U2 snRNP, which interacts with the branch site.

Spliceosome Cycle Steps 4-5 & 7

• Formation of the B-complex with the addition of U4/U6 and U5 snRNPs facilitates further processing of the spliceosome.
• Splicing catalysis occurs in the C-complex, where U6 snRNP pairing initiates the bond formation, U2 snRNP pairing enables the branch site, and U5 snRNP interaction connects the exons.
• Cycle concludes with the release of the snRNPs for reuse in future splicing events.

Different Types of RNA Splicing

• Nuclear RNA encoding proteins (cis-splicing) requires conserved sequences in introns and spliceosomes.
• Trans-splicing of nuclear RNA involves the splicing of separate pre-mRNA molecules.
• Self-splicing (Type I/II) occurs through intron folding and interactions, and is independent of proteins.
• tRNA and rRNA undergo special splicing mechanisms.

Trans-Splicing of Nuclear RNAs

• The product is a chimeric mRNA molecule assembled from two different transcripts.
• Trans-splicing is a rare but important process with roles in gene expression that generate great diversity.

Production of rRNAs

• rRNA synthesis involves multiple steps, not true splicing.
• The large and smaller rRNAs are created from a precursor rRNA by cleavage.

rRNA Processing by snoRNAs

• snoRNAs, a class of small RNA molecules, influence rRNA modification.
• Pre-rRNA undergoes cleavage, methylation, and other modifications assisted by snoRNAs.
• Cleavage, nucleases (various types) involvement is key for rRNA processing.

Other Variants of Introns: U12-Dependent Introns

• Alternative splicing mechanism involving U12 snRNPs.
• Minor splicing class has AT-AC rules associated with it.
• Involves a unique set of factors like U12 snRNP.

tRNA Processing by RNases and Splicing

• tRNA processing uses specific enzymes and depends on the tRNA's secondary structure.
• An endonuclease, acting at the intron's edges in the mature tRNA, catalyzes a splicing-like event.
• Involves RNases and other enzymes to create mature tRNA, with specific base modification in the intermediate stage.