Class 9 transcript DS

RNA Polymerase II

1. Enzyme that transcribes mRNA,

2. contains C-terminal domain (CTD) that coordinates processing events through phosphorylation changes

CTD Phosphorylation what is it, describe it different phases of transcription

Key regulatory mechanism:

Ser5 phosphorylation during initiation recruits capping proteins,

Ser2 phosphorylation during elongation recruits splicing proteins,

dephosphorylation near termination allows poly-A tail addition

5' Cap, what is it, what does it do, when is it added, etc

Modified guanine nucleotide added to beginning of mRNA,

protects from exonucleases,

recognized by translation machinery,
added during transcription initiation

3' Poly-A Tail what is it, what does it do, when is it added

Adenine nucleotides added after cleavage at poly-A signal,

protects mRNA from degradation,

facilitates nuclear export and translation,

added during termination phase, after cleavage

Splicing, what is it, when does it occur,…

Process of removing introns and joining exons in mRNA,

occurs co-transcriptionally during elongation,

recognizes specific splice site sequences

Constitutive Splicing

Default splicing pattern where all introns are removed and all exons are joined in their genomic order

Alternative Splicing

Deviation from constitutive splicing that produces different mRNA isoforms from the same gene through various mechanisms

Exon Skipping

Type of alternative splicing where certain exons are omitted from the final mRNA transcript

Intron Retention

Type of alternative splicing where certain introns are not removed and remain in the mature mRNA

Alternative 5' Splice Site

Splicing variation that changes the upstream exon length by using different donor splice sites

Alternative 3' Splice Site

Splicing variation that changes the downstream exon length by using different acceptor splice sites

Alternative Transcriptional Start Site

Mechanism producing different mRNA isoforms by initiating transcription at different positions within a gene

snRNPs (snurps)

1. RNA-protein complexes that

2. recognize splice sites through base pairing between snRNA and mRNA consensus sequences

Splice Site Consensus Sequences

Specific nucleotide motifs recognized by splicing machinery: 5' splice site, 3' splice site, and branch point sequences

Splicing Enhancers

Regulatory sequences and binding proteins that promote splicing at specific sites

Splicing Silencers

Regulatory sequences and binding proteins that inhibit splicing at specific sites

RNA Secondary Structure

mRNA folding through base pairing that can block splice site accessibility and regulate alternative splicing

Chromatin Regulation of Splicing

Histone modifications and DNA methylation that can influence splicing factor recruitment and alternative splicing patterns

RNA Sequencing (RNA-seq)

Method to detect alternative splicing by sequencing all transcripts, reveals different mRNA isoforms and their abundances

RT-PCR

Reverse transcription PCR using RNA template to detect and quantify specific spliced variants, requires prior knowledge of target sequences

Northern Blot

Technique separating RNA by gel electrophoresis, transferring to membrane, and probing with labeled DNA to detect specific transcripts and their sizes

Exam Reflection Assignment

Required self-assessment of exam performance and study habits, prerequisite for submitting regrade requests

Regrade Request Process

Opportunity to request exam regrading available after completing exam reflection, due within specific timeframe

Office Hours Policy

Students can attend any professor's office hours regardless of who is teaching particular unit

Unit 2 Transition

Moving from transcription-focused unit to translation-focused unit, emphasizing various RNA types and processing steps

Learning Objectives

Posted guidelines for unit content, intended to guide but not limit study focus to exam material only

Dscam Gene

Famous example of extreme alternative splicing with thousands of variants, involved in immunity and neuronal development

Co-transcriptional Processing

Concept that mRNA capping, splicing, and polyadenylation occur simultaneously with transcription rather than as separate steps

Untranslated Regions (UTRs)

Portions of exons that are not translated into protein but contain regulatory elements for translation and stability

Exon Definition

Any sequence retained in the final mature mRNA is considered an exon, regardless of protein-coding status

Branch Point

Adenine nucleotide within intron that participates in the first step of splicing through 2'-5' phosphodiester bond formation

Spliceosome

Large ribonucleoprotein complex composed of snRNPs that catalyzes the splicing reaction through RNA-RNA and RNA-protein interactions

Consensus Sequence Motifs

Characteristic nucleotide patterns that define functional elements like splice sites, recognized by complementary base pairing

Transcript Variants

Different mRNA isoforms produced from the same gene through alternative splicing, alternative start sites, or other processing variations

RNA-Protein Interactions

Fundamental feature of molecular biology where RNA molecules interact with proteins to carry out processing and regulatory functions

Base Pairing Rules

Fundamental principle where complementary nucleotide sequences allow specific recognition in splicing and other RNA processing events

Gel Electrophoresis Prediction

Method to distinguish splicing variants: exon skipping produces smaller products, intron retention produces larger products

Sequence-Specific Recognition

Principle that splicing machinery identifies sites through complementary base pairing with consensus sequences in the RNA

Phosphorylation State Changes

Dynamic modifications of RNA polymerase II CTD that coordinate the timing of different processing events during transcription

Protection from Nucleases

Primary function of 5' cap and 3' poly-A tail in preventing degradation of mRNA ends by cellular exonucleases

Nuclear Export Requirement

Poly-A tail is necessary for mRNA to be transported from nucleus to cytoplasm for translation

Cleavage Preceding Polyadenylation

Process where mRNA is first cleaved at poly-A signal site before poly-A polymerase adds the tail

Splicing Regulation Mechanisms

Three main types: protein binding to enhancer/silencer sequences, RNA secondary structure blocking sites, chromatin modifications influencing factor recruitment

Experimental Size Analysis

Using techniques like northern blot or gel electrophoresis to compare transcript sizes and infer splicing events based on migration patterns

mRNA Stability

Property influenced by 5' cap, 3' poly-A tail, and sequence elements that determine how long mRNA persists in the cell before degradation

Translation Machinery Recognition

Process where ribosomes identify start codons within mRNA context, typically internal to the transcript rather than at exact ends

Molecular Biology Emphasis

Unique aspect of this field featuring extensive RNA-protein interactions and RNA-based regulatory mechanisms compared to other biological disciplines

5' Splice Site Consensus

GU-rich sequence at 5' end of intron, recognized by U1 snRNP through complementary base pairing

3' Splice Site Consensus

AG-rich sequence at 3' end of intron, recognized by U2AF and other splicing factors

Branch Point Sequence

YNYURAC (where Y=pyrimidine, R=purine) located 20-50 nucleotides upstream of 3' splice site, adenosine serves as nucleophile

Poly-A Signal

AAUAAA sequence recognized by cleavage and polyadenylation specificity factor (CPSF)

7-methylguanosine Cap

Modified guanine nucleotide connected via 5'-5' triphosphate linkage, added by capping enzymes early in transcription

U1 snRNP

Recognizes and binds to 5' splice site through RNA-RNA base pairing between U1 snRNA and consensus sequence

U2 snRNP

Binds to branch point sequence, displacing the adenosine to make it reactive for first transesterification

U4/U6.U5 Tri-snRNP

Complex that undergoes conformational changes and catalytic activation during spliceosome assembly

First Transesterification

Branch point adenosine attacks 5' splice site, creating 2'-5' phosphodiester bond and lariat structure

Second Transesterification

3' OH of freed exon attacks 3' splice site, joining exons and releasing intron lariat

Lariat Structure

Circular RNA structure formed when 5' end of intron joins to branch point adenosine via 2'-5' bond

Ser5 Phosphorylation

Occurs during initiation phase, recruits capping enzymes to CTD of RNA polymerase II

Ser2 Phosphorylation

Occurs during elongation phase, recruits splicing machinery to CTD

RNA-seq Advantage

Can detect novel splicing variants without prior knowledge of specific isoforms

Northern Blot Transfer

RNA separated by gel electrophoresis is transferred to nylon or nitrocellulose membrane for probing

Membrane Probing

Labeled DNA complementary to target sequence hybridizes to specific RNAs on northern blot membrane

Size Determination

Comparing migration distances on gel to known standards identifies transcript sizes and splicing variants

In-class Gene Example

Featured exons colored: red, yellow, blue, gray with orange introns (specific gene name not given)

Variant 1 Analysis

Orange intron retention - larger transcript size on northern blot

Variant 2 Analysis

Alternative 5' or 3' splice site usage - intermediate transcript size, requires sequencing to distinguish

Variant 3 Analysis

Yellow exon skipping - smaller transcript size

Variant 4 Analysis

Alternative transcriptional start site in orange region - distinct isoform pattern

Dscam Protein Function

Involved in neuronal wiring and immune recognition through extensive alternative splicing diversity

Chromatin Influence

Histone modifications can recruit splicing factors to specific genomic regions, affecting splice site choice

RNA Structure Blocking

Hairpins and stem-loops can physically prevent spliceosome access to splice sites

Splicing Regulatory Proteins

Bind to exonic/intronic enhancer/silencer elements to promote or inhibit splice site usage

Co-transcriptional Timing

Capping occurs immediately after 5' end emergence, splicing during elongation, polyadenylation after cleavage

CTD as Recruitment Platform

Phosphorylated C-terminal domain serves as binding site for processing factors throughout transcription cycle

Untranslated Regions Importance

5' UTR contains ribosome binding sites, 3' UTR has stability and localization signals

Exon Definition Principle

Any sequence retained in mature mRNA qualifies as exon, regardless of coding potential

Spliceosome Assembly

Stepwise process involving U1, U2, and U4/U6.U5 snRNPs with extensive RNA-RNA rearrangements

Catalytic RNA Role

snRNAs (not proteins) provide the active site for transesterification reactions in splicing

Alternative Start Sites

Can produce isoforms with different first exons, affecting translation efficiency and regulation

Cell Type Specific Splicing

Different tissues express distinct splicing regulator proteins, creating tissue-specific isoform patterns