mRNA Splicing - mRNA post-transcriptional processing/modifications - What is alternative splicing?

What is Splicing?

General Definition and Overview

• Splicing is the process of removing introns and, in some cases, exons from pre-mRNA (PMRNA) during or after transcription.

• The video will be divided into three parts: what splicing is, how it occurs, and why it is necessary.

Structure of Pre-mRNA

• A simple PMRNA structure consists of two exons (E1 and E2) separated by an intron (I). The focus will be on the intron syntax.

• Intron syntax includes specific nucleotide sequences at both ends: a guanine-uracil (GU) pair at the 5' splice site and adenine-rich sequences towards the end.

Intron Syntax Details

Key Components of Intron Syntax

• The five prime splice site has a consistent presence of GU nucleotides; this is crucial for splicing initiation.

• The branch point within the intron contains adenines, followed by a pyrimidine track rich in U's and C's leading to the three prime splice site enriched with adenine and guanine.

Types of Introns

• Approximately 90% of PMRNAs contain U2 type introns characterized by specific splice sites; remaining 10% are U12 type with different nucleotide compositions at their splice sites.

Types of Introns Beyond Pre-mRNA

Classification of Introns

• There are various types of introns including nuclear pre-mRNA (U2 & U12), organelle-specific introns, and bacterial introns which can be self-splicing or require endonucleases for removal.

• Self-splicing introns act as ribozymes that do not need proteins for their removal; they are found in mRNAs, tRNAs, and rRNAs across different organisms.

Spliceosome Functionality

Role in Splicing Process

• Focus will remain on splicosome-dependent U2 type introns where splicing begins once the splicosome proteins recognize the intron syntax.

Composition of Spliceosomal Proteins

• Small nuclear ribonucleoproteins (snRNPs), composed of small nuclear RNA (snRNA) and protein components, play a critical role in recognizing these signals during splicing processes. Major snRNP types include U1, U2, U4, U5, and U6.

Mechanism of Splicing

How Splicing Occurs

• The process involves complex interactions among snRNP components that form secondary structures essential for recognizing splice sites within the PMRNA strand containing a U2 type intron.

Splicing Mechanism Overview

Recognition Stage of Splicing

• The recognition begins at the five-prime splice site where U1 snRNP pairs with GU in pre-mRNA, marking the initial step in splicing.

• SR proteins, characterized by serine and arginine repeats, stabilize U1 snRNP binding to RNA and assist in splicing regulation.

• The branch point adenine is recognized by splicing factor 1 (SF1), while U2 AF protein dimer binds to the three-prime splice site, enhancing recognition through cooperative binding.

• This recognition occurs in a looped conformation due to long introns, facilitating interactions between U1, SF1, and U2 AF proteins.

• The early complex forms as SR proteins help bring exons closer together; this complex is also known as a stable or committed complex.

Formation of Pre-catalytic Complexes

• After the early complex assembles, U2 snRNP replaces SF1 and U2 AF proteins at the branch point, forming the A complex while maintaining U1 at the five-prime splice site.

• The B1 complex emerges when U5, U4, and U6 join; here, U5 bridges exons while replacing SR proteins' function within the assembly.

• To activate catalysis, both U1 and U4 are removed from this assembly; thus creating a B2 complex that is catalytically active for splicing reactions.

Catalytic Steps of Splicing

• The first transesterification reaction occurs when hydroxyl from adenine attacks phosphate at the five-prime splice site; this cleaves off exon from intron.

• As exons are cut free from intron lariat structure remains bound to snRNP complexes; this leads to formation of catalytic one complex.

• In the second transesterification step, upstream exon attacks three-prime splice site leading to ligation of two exons while releasing intron lariat structure.

• The resulting structure post-ligation shows two joined exons with an attached lariat-shaped intron still associated with snRNP complexes.

• Special RNA helicases facilitate removal of SF1 and other components during these steps which are crucial for successful splicing.

Detailed Mechanism Insights

• Examination reveals phosphodiester linkages between G and U at five-prime splice site alongside sugar-phosphate connections around branch point adenine during transesterification steps.

• First transesterification involves attack by two-prime hydroxyl on guanosine phosphate leading to formation of looped intron lariat structure observed in catalytic one complex.

• Second step sees upstream exon’s free hydroxyl group attacking three-prime splice site phosphate which releases intron lariat allowing mRNA formation through exon joining.

Understanding Splicing Mechanisms

The Basics of Splicing

• Splicing is crucial for removing introns and joining exons in mRNA, which is essential for producing functional proteins.

• Canonical splicing involves the independent removal of introns and the joining of exons, allowing for various combinations to form different mRNAs.

• The removal of introns prevents the production of defective proteins since introns are non-coding regions. This process enhances protein diversity through alternative splicing.

Alternative Splicing Explained

• Alternative splicing allows a single gene to produce multiple RNA isoforms by varying exon combinations, significantly increasing protein diversity within cells.

• To achieve specific exon arrangements (e.g., E1 and E3), certain proteins must prevent other exons from participating in splicing, showcasing the complexity of this regulatory mechanism.

Trans Splicing in Worms

• In model organisms like worms, trans splicing occurs where exons from two different RNAs are joined together, involving unique elements called outrons.

• The splice leader RNA (SLRNA), containing a short exon and an intron, plays a key role in trans splicing by transferring important features like the 5' cap to stabilize mRNA.

Understanding Outrons

• During trans splicing, the SLRNA's branch point interacts with the PMRNA's splice site to replace its 5' UTR with that from SLRNA, enhancing stability.

• Outrons differ from traditional introns; their removal results in a branched lariat structure distinct from typical intron lariats observed during canonical splicing.

Back Splicing: A Unique Process

• Back splicing involves an exon attacking its own previous end rather than following standard forward directionality during canonical splicing.

• This type of splicing can lead to circular RNA formation and is often associated with pathological conditions rather than normal cellular processes.