Transcriptional elongation

What direction is DNA transcribed in?

read in the 3’ → 5’ direction, mRNA synthesized in 5’ → 3’ direction

Which strand of DNA is transcribed?

The template, antisense, non-coding strand

Which strand of DNA resembles the mRNA?

The sense, coding strand

What is a transcription bubble?

RNA Polymerase II pulls apart the DNA into single strands in a small region, called the transcription bubble. Once RNA polymerase II leaves a region, the DNA comes back together into double strands

What is the RNA-DNA hybrid?

This is a transient hybrid that forms between the newly synthesized RNA and the template strand of the DNA during transcription. The complementary base pairs hydrogen bond with each other within the RNA Pol II complex. This hybrid is about 8 bp long and is quite strong, but again is transient so it does not stay intact for long

What is early elongation?

RNA Pol II “escapes” from the PIC and starts transcription for about 20-60 nucleotides and then it pauses, sometimes it has not even reached the coding sequence of the gene yet

What are some reasons why RNA Pol II is believed to undergo pausing?

• some motifs in downstream region of promoter are very GC-rich and can form very strong RNA-DNA hybrids with the newly synthesized RNA, and this prevents the RNA Pol II from moving forward along the gene

• Mediator complex, NELF, and DSIF contribute to pausing → NELF and DSIF bind directly to RNA Pol II

How is pausing of RNA Pol II alleviated?

• phosphorylation

• signals for alleviation of pausing are often intercellular signals → ex. growth factor leads to phosphorylation cascade

• recruitment of P-TEFb (positive transcription elongation factor b) to Pol II, which contains a kinase that phosphorylates RNA Pol II, NELF, and DSIF

• this releases NELF and DSIF from RNA Pol II and allows RNA Pol II to continue transcription

What are benefits of RNA Pol II pausing?

• RNA Pol II association with DNA lasts longer when it is paused during transcription (~10 minutes) versus when it is just associated with the PIC and promoter (a few seconds)

• This can poise genes for rapid activation → instead of having to assemble all parts of PIC, mediator complex, RNA Pol II, and all other necessary factors for activation of transcription of gene, if RNA Pol II is already on the gene and just paused, it is a lot faster to activate that transcription

• Can be useful for pathways that have complex interactions and need to be highly coordinated

Does every gene in the genome exhibit RNA Pol II pausing? What stages of a cell’s life exhibit more RNA Pol II pausing?

Not every gene in the genome exhibits RNA Pol II pausing, and not every gene will continuously exhibit RNA Pol II pausing even if it has at one point; RNA Pol II pausing is predominant during development stages of life or regeneration stages

Which promoter motif is associated predominantly with RNA Pol II pausing?

the initiator motif that overlaps with the TSS

what happened when people made mutants of NELF that prevented RNA Pol II from occurring?

It was early embryo lethal, so the mutants died

What happens after RNA Pol II is released from pausing?

Productive elongation → association of addition elongation factors with RNA Pol II like ELL and FACT; these keep RNA Pol II stable and deal with issues in chromatin state

can multiple RNA Pol II bind to a gene at a time?

Yes, as one moves down the gene, another one can bind to a new PIC and so on

How does RNA Pol II pausing keep the chromatin state open?

Because if RNA Pol II is bound and paused, this blocks the region from being silenced by molecules like DNA methyltransferases

Does transcription occur continuously?

No, it occurs in bursts where many RNA Pol II molecules may bind to the gene and transcribe it and then there will be inactive periods where no RNA Pol II molecules are bound to the gene

How was it observed that transcription occurs in bursts?

Add to gene of interest a gene for MS2 loops. As the transcript is being made the RNA forms loops, which can be recognized by MCP. GFP (green fluorescent protein) can be bound to MCP so that the loops become visible when MCP-GFP attached to them. They then used a microscope to see the fluorescence intensity and then found that the intensity fluctuates over time, indicating bursts of transcription

Do all genes have the same quantity/quality of bursting?

No

• the core promoter of the gene determines the burst size (how many transcribing RNA Pol II molecules can bind)

• the enhancers (cis-regulatory elements) regulate the burst frequency (how long the “on” and “off” periods of transcription are)

Does processing of newly synthesized mRNA occur during transcription or after it is completed?

It occurs while the mRNA is still being synthesized

What is a 5’ cap and what is its purpose?

While RNA is being synthesized, a methyl-guanine cap is added on to the 5’ end of the RNA in the reverse orientation (5’ end of methyl-guanine is connected to the 5’ end of the mRNA through a triphosphate bridge). The guanine is added by guanylyl-transferase and the methyl group is added to the guanine by guanine-7-methyltransferase

• The cap protects the transcript from degradation by RNases

• The cap recruits cap-binding proteins, which are necessary to transport the processed mRNA out of the nucleus and into the cytoplasm for translation

• In the cytoplasm, the cap recruits other proteins necessary for translation (eukaryotic elongation factors and RNA helicase); these proteins also recognized poly-A binding proteins to form a pseudocircular structure that doesn’t have free ends; this protects the mRNA from nucleases that break down RNAs with free ends (like those from RNA viruses)

where does splicing occur in mRNA transcripts?

It occurs at conserved GU-AG splice sites with intervening branch sites; the GU is the splice donor sequence and the AG is the splice acceptor sequence; the branch site within the intron is usually sequence ACT (not always) and this is what the splicing factors recognize when it is time to remove the introns; proteins recognize these splice donor and acceptor sites and remove the introns

Are the nucleotides around the splice donor and acceptor sites random?

No, there are biases for certain nucleotides near the splice sites. For example, there is usually a G in the exon next to the GU splice donor site. This implies that there may be some function importance of the nucleotides around splice sites because there has been selection to maintain those certain nucleotides

Are the ends of exons always the ends of codons?

No, codons can be split across exons, so the full codon isn’t next to each other until splicing out of introns occurs

How are the introns actually removed from pre-mRNA?

They are cut out by spliceosomes, which are small nuclear ribonucleoprotein complexes comprised of one small RNA (U1, U2, U5, U4/U6) and ~20 proteins; the spliceosomes associate with the pre-mRNA as it is still being transcribed and recognize the specific splice donor and acceptor sites with the help of other proteins; first, there is a cut at the 5’ splice donor site, forming a loop (lariat) which folds back on the branch site in the intron, forming a 5’-2’ bond; then, the splice acceptor site is cleaved and the removed intron goes off to get degraded and the 5’ and 3’ sites of the exon are spliced together, forming a new phosphodiester bond

What usually causes errors in splicing?

Genetic mutations

What is intermediate filament Lamin A (LMNA) and what are some examples of splicing errors in this gene and their results?

LMNA is a component of the nuclear lamina and is required for structural stability, nuclear organization, cell motility, mechanosensing, etc. Different mutations in the LMNA gene result in abnormal splicing and thus different phenotypes and pathologies

• In Limb Girdle muscular dystrophy 1B, a G within an intron gets changed to a C, resulting in the retention of the intron in the final mRNA; the presence of the intron in the final transcript results in a premature stop codon and therefore a truncated protein

• A similar thing occurs in Familial partial lipodystrophy type 2, where a G within a different intron gets changed to a C and the intron is not removed and it has a premature stop codon in it leading to truncated protein

• In Hutchinson-Gilford progeria, a C in an exon gets changed to a T, resulting in deletion of some exon sequence, resulting in a smaller protein that is missing some amino acids

• In Dilated cardiomyopathy, an A in an intron gets changed to a G, resulting in retention of an intron as coding sequence and extra amino acids in the protein

Is it easy to predict the outcome of a given mutation on splicing outcome?

No, it is very difficult to predict the effect a mutation will have on splicing

What are the most important sites in an intron?

The two nucleotides at the 5’ end (splice donor site) and the two nucleotides at the 3’ end (splice acceptor site)

Does all alternative splicing lead to pathologies?

No, there is a considerable amount of normal alternative splicing in the genome

Do all genes only code for one protein?

No, almost all genes code for multiple proteins, indicating many splice variants

What are ways that variable mRNA transcripts can be obtained from a single gene?

• Variation in core promoters and transcription start sites → transcription can start at different places in the gene

• Variation in transcription termination sites or polyadenylation → transcription ends at different sites in the 3’ UTR

• Variation in splicing to retain different exons or introns → produce splice variants

How common is alternative splicing?

Very common; 95-100% of pre-mRNAs with more than one exon yield multiple mRNAs; there can be 2 - 1000s of variants per gene

What is an example or normal alternative splicing?

• Dscam pre-mRNA → codes for cell adhesion molecule in nervous system; Exons 4, 6, and 9 have many potential possible exons within them, but only one from each exon ends up in the final mRNA, and this generates tens of thousands of different possible mRNAs; this generates tremendous specificity in the nervous system because it dictates which neurons will connect to one another

• mod(mdg4) gene → there are many alternative terminal exons, which can determines how the protein interacts with other proteins and molecules

What are two mechanisms of alternative splicing?

1. More direct method: cis and trans splicing regulators that influence spliceosome binding/activity → cis regulators are mRNA motifs present in mRNA → exonic/intronic splicing enhancers/suppressors (ex. ESE); trans regulators are proteins that bind to cis motifs → serine/arginine (SR) rich proteins promote splice site usage and compete with repressive, heterogenous nuclear ribonucleoproteins (hnRNPs) and tissue specific regulators (different abundance in different cells)

2. Less direct method: Transcription rate differentially exposes exons to splicing factors → histone methylation/acetylation influences RNA Pol II speed based on the state of the chromatin (more open chromatin → faster RNA Pol II); if RNA Pol II is too fast, it can make RNA faster than the spliceosome can process it, making it more prone to errors in splicing; if RNA Pol II is moving slower, the spliceosome can keep up and process the RNA with more certainty; this means that precise histone modification and chromatin state may favor some splice variants over others

Which cis motifs would SR proteins bind to?

Exonic splicing enhacers (ESEs) or Intronic splicing enhancers (ISEs)

Which cis motifs would hnRNPs bind to ?

Exonic splicing supressors (ESSs) or intronic splicing suppressors (ISSs)

are the significance of most normal splice variant protein isoforms known?

No, unless there is a bonafide pathology there

What is Titin?

It is the longest protein known, at about one micron long; it regulates the length and elasticity of striated muscle; alternative splicing of Titin between fetuses and adults regulates cardiac muscle elasticity, and this splicing depends on splicing factor RBM20

What regulates the elasticity of Titin?

If certain exons are spliced out, it will be less elastic; if they are retained it will be more elastic; the more elastic variant is present in fetuses, and the less elastic variant is present in adults; the transition to the less elastic variant is due to RBM20

What occurs if RBM20 does not function properly?

There is no transition from more elastic Titin to less elastic Titin, and this retainment of elastic Titin results in fibrotic cardiomyopathy, which can be fatal

What modification to the 3’ end of pre-mRNA occurs once transcription is complete?

poly-adenylation (-AAAAAAAAA)

Are transcription termination signals invariant?

No, there is a lot of variability in when transcription ends

How does polyadenylation occur?

RNA-Binding Proteins (RBPs) associated with RNA Pol II and recognize poly-adenylation (and other) motifs in the 3’ UTR of the newly synthesized mRNA; this complex cleaves the mRNA at the 3’ Poly-A site and recrutis Poly-A Polymerase (PAP), which will add the poly-A tail to the 3’ end of the mRNA

Is the poly-A tail encoded in the gene?

No, it gets added on after transcription by PAP

What is the purpose of the poly-A tail on mRNA?

• helps get mRNA into translatable configuration for translation

• allows for recruitment of poly-A binding proteins in nucleus and cytosol (PABPN and PABPC) → as the poly-A tail is formed, it gets decorated with these PABPs

What are the functions of PABPN and PABPC?

• PABPN → decorates poly-A tail while it’s being added; promotes PAP activity, allows loop to be formed by interacting with Cleavage and Polyadenylation Factor (CPSF) at beginning of poly-A tail (this influences the total length of the poly-A tail), functions in RNA export from nucleus

• PABPC → associates with poly-A tail in cytosol; interact with other RBPs (eukaryotic initiation factor proteins, eIFs) to allow loop of mRNA to be formed, forming bridge between 5’ end of mRNA and 3’ end

What molecules/regions of molecules do eukaryotic initiation factor proteins (eIFs) interact with?

PABPCs on poly-A tail, 5’ end of mRNA, or small subunit of ribosomes

What is the purpose of the mRNA loop formed in the cytosol by PABPC and eIFs?

it helps to recruit the full ribosome and it protects the mRNA from exonucleases that will chew it up because they target mRNAs with free ends

What would the result of a shorter poly-A tail be?

Limited opportunity for PABPC binding, less translational efficiency, and less protection from exonucleases