advanced exam 2 L10-L11

RNA transcription, RNA processing, FSHD

What is the central dogma?

DNA encodes RNA, which encodes protein. Genomic DNA is the template for RNA synthesis (transcription), and RNA is decoded into protein (translation).

Which RNAs are translated into protein, and what share of total cellular RNA are they?

Only mRNA is translated. mRNA is about 3-5% of total cellular RNA; most RNAs are never translated and have other roles.

How does RNA differ from DNA structurally?

RNA has ribose (not 2'-deoxyribose) and uracil in place of thymine. Purines (A, G) and cytosine are the same. A pairs with U through the same hydrogen-bonding groups used in A-T pairing.

In which direction is RNA synthesized, and is a primer needed?

RNA is synthesized 5' to 3' using DNA as a template. No primer is required for initiation.

Which DNA strand has the same sequence as the RNA transcript?

The sense (coding) strand has the same sequence as the RNA (with U in place of T). The antisense strand is the transcribed strand and serves as the template.

What do promoters do, and where are they located?

specific DNA sequences upstream (5') of the coding sequence that mark where transcription begins. They give specificity and help determine how much RNA (and therefore protein) is made.

Why is transcription an important step in gene regulation?

Genes are transcribed with different efficiencies. If gene A is transcribed much more than gene B, more gene A product (protein) is made.

What are the core bacterial promoter elements?

The TATA box at -10 (a TA-rich region that is easier to melt than GC-rich DNA) and the TTGACA sequence at -35. Variation in these sequences changes expression, and mutations reduce transcription.

What does RNA polymerase bind in bacteria?

Promoter sequences (the -10 and -35 elements).

Why is transcription in eukaryotes not coupled to translation?

Transcription occurs in the nucleus where the DNA is, while ribosomes are in the cytoplasm. In bacteria there is no compartmentalization, so translation can begin before transcription finishes.

What extra requirements does eukaryotic transcription have compared with bacterial?

Chromatin remodeling (unwinding DNA, moving histones), and the RNA must be processed (splicing, capping, etc.) to make mature mRNA.

How many RNA polymerases do eukaryotes have, and which makes mRNA?

Three. RNA polymerase II (RNAP II) makes mRNA.

What does RNA polymerase I transcribe?

The 5.8S, 18S and 28S rRNA genes.

What does RNA polymerase II transcribe?

All protein-coding genes, plus snoRNA, miRNA, siRNA and lncRNA genes and most snRNA genes.

What does RNA polymerase III transcribe?

tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs.

What does the "S" in rRNA names (e.g. 18S) refer to?

The sedimentation rate in an ultracentrifuge. A larger S value means a larger rRNA.

What elements make up the eukaryotic RNAP II core promoter?

The TATA box (about -30 in some genes; weaker AT pairing helps open the helix), BRE (B recognition element, about -35, binds TFIIB), INR (initiator, can substitute for or combine with TATA) and DPE (downstream promoter element, found in few promoters). Not every promoter has all of them, but some element is required.

What does the BRE bind?

TFIIB (a transcription factor).

What is the INR element?

The initiator element. It can substitute for the TATA box or combine with it to increase efficiency.

Define cis-acting vs trans-acting in transcription.

Cis-acting: DNA sequences on the strand being copied. Trans-acting: proteins from outside (e.g. transcription factors).

Why can't RNAP II bind DNA directly?

It needs general transcription factors to position it correctly at the promoter, pull the DNA strands apart, and release it to begin elongation.

What do specific transcription factors do?

They influence the rate and efficiency of transcription and can enhance transcription in certain cell types and conditions.

What does TFIID do?

12 subunits. Recognizes the TATA box and other sequences near the start point (via the TATA binding protein, TBP, one of its components).

What does TFIIB do?

1 subunit. Recognizes the BRE and accurately positions RNA polymerase at the transcription start site.

What does TFIIA do?

2 subunits. Not required at all promoters; stabilizes TFIID binding.

What does TFIIF do?

3 subunits. Stabilizes RNA polymerase interaction with TFIIB and helps attract TFIIE and TFIIH.

What does TFIIE do?

2 subunits. Attracts and regulates TFIIH.

What are the functions of TFIIH?

10 subunits. It is a helicase that uses ATP to unwind DNA at the start point, and a kinase that phosphorylates Ser5 of the RNAP II C-terminal domain (CTD), which releases the polymerase from the promoter.

What are the two functional domains of a transcription factor?

A DNA-binding domain and a trans-activating domain (affects transcription through protein-protein interaction).

What is TBP?

TATA binding protein; a component of TFIID that binds the TATA sequence in the promoter.

Order the steps of eukaryotic transcription initiation.

1) TFIID (TBP) binds the TATA box. 2) TFIID binding distorts the DNA, allowing further assembly. 3) Other factors and RNA pol II join to complete the initiation complex. 4) TFIIH hydrolyzes ATP to unwind the DNA (helicase) and phosphorylates the CTD (kinase). 5) The phosphorylated polymerase releases the transcription factors and begins elongation.

What is the role of mediator?

Mediator links activators to the polymerase. It does not need to be close to the gene and can loop DNA to activate transcription from afar.

What does CTD phosphorylation accomplish?

It lets RNAP II leave the promoter and elongate. General factors are released and can start another round. The phosphorylated CTD also attracts RNA processing factors that modify the RNA as it emerges.

What is an enhancer?

A DNA sequence (upstream, within or downstream of a gene) bound by activator proteins that modulates transcription from a distance.

What is a silencer?

A DNA sequence that binds proteins that repress expression.

How can a gene be expressed in only one cell type?

Its promoter has sequences recognized by transcription factors unique to that cell type. Alternatively, the factors may be present in all cells but only activated in one.

What is a pre-mRNA?

The unmodified primary transcript made by RNA polymerase II. It becomes mature mRNA only after processing.

List the three RNA processing reactions for pre-mRNA.

5' capping, splicing (removing introns), and 3' cleavage and polyadenylation.

When does capping occur, and why is it needed?

As soon as the RNA is about 20 nt long. The cap marks the transcript as mRNA and is required for mRNA stability, export from the nucleus, and recruitment to ribosomes for translation.

What three enzymes carry out capping?

Phosphatase (removes one phosphate from the 5' end), guanyl transferase (adds GMP via a 5'-5' linkage), and methyl transferase (adds a methyl group to the 7 position of the guanosine).

What is the structure of the 5' cap?

7-methylguanosine joined to the first nucleotide by an unusual 5'-5' triphosphate linkage: meGpppN.

What does the cap binding complex (CBC) do?

It stays bound to the cap, protects the RNA, assists in processing and export, and is displaced by translation initiation factors.

Which genes lack introns?

Histone genes and interferon genes. Most other eukaryotic genes have at least some introns.

Is intron number or size related to gene size?

No. The size and number of introns and the size of the gene are not related.

When does splicing occur relative to transcription?

During transcription. Part of the splicing machinery loads onto the RNAP II complex, so splicing is coupled to transcription.

How big is the splicing machinery?

Over 50 proteins and 5 RNA molecules. The RNAs act like enzymes.

Describe the two transesterification reactions of splicing.

1) A specific adenine (the branch point A) in the intron attacks the 5' splice site and cuts the backbone, forming a lariat. 2) The 3'-OH of the first exon attacks the start of the next exon, joining the exons and releasing the intron as a lariat. The lariat is then degraded to single nucleotides.

What sequence elements define an intron?

5' end: exon ends in MAG (M = A or C) and intron begins with GURAG (R = A or G). 3' end: intron ends in CAG, exon begins with G. A polypyrimidine tract lies upstream of the 3' splice site, and the branch-point A lies upstream of that.

Does splicing require ATP?

Yes. Intron excision requires ATP hydrolysis and is highly accurate.

Which snRNAs take part in splicing?

U1, U2, U4, U5 and U6 (100-200 nt each). U3 is involved in rRNA processing, not splicing.

What is an snRNP, and what do they form?

A small nuclear ribonucleoprotein: an snRNA complexed with many proteins. snRNPs form the spliceosome.

Why does the spliceosome count as an RNA enzyme?

Its active site contains almost exclusively RNA components (the snRNAs), so catalysis of cleavage and rejoining is by RNA, not protein.

How does the spliceosome check intron sequences for errors?

Conformational changes shift base-pairing interactions from one position to another, giving error checking before cleavage occurs.

Spliceosome step 1: what do U1, U2AF and BBP recognize?

U1 snRNP base-pairs with the 5' splice junction. U2AF recognizes the polypyrimidine tract and 3' splice junction. BBP (branch-point binding protein) recognizes the branch-point site and binds cooperatively with U2AF.

Spliceosome step 2: what does U2 snRNP do?

It displaces BBP and U2AF and base-pairs with the branch-point consensus sequence, freeing the branch-point adenine to attack the 5' splice junction.

Spliceosome steps 3-4: what is the U4/U6+U5 triple snRNP and what happens to it?

U4 and U6 are held together by base pairs and enter with U5. Rearrangements break the U4/U6 pairs, U6 displaces U1 at the 5' splice junction, and U4 and some U6 proteins are ejected.

Spliceosome step 5: how is the active site formed?

The NTC/NTR protein complex positions the snRNPs. U2 and U6 RNAs bind each other and bind the RNA at different places, bringing the intron/exon junctions together and the branch point near the 5' splice site.

What is the exon junction complex (EJC)?

A set of proteins that binds the completed splice junction, marks splicing as complete, and stays on the mRNA.

What happens at the end of splicing?

The spliced RNA is released, the intron leaves as a lariat still bound to U2, U5 and U6, and spliceosome components are recycled using ATP hydrolysis.

What are self-splicing introns and where are they found?

Introns that splice without any proteins or additional RNAs. They are found mainly in rRNA and organellar (mitochondrial) transcripts, and support the idea that early life was RNA-based.

What is exon definition?

Introns vary hugely in size (over 100,000 nt) but exons are consistently about 150 nt. SR proteins bind exons during transcription and mark their boundaries, which helps splicing accuracy and regulation.

Why does coupling splicing to transcription reduce mistakes?

snRNPs at the 5' splice site only see the 3' splice site that emerges next from the polymerase.

Name three ways a single-nucleotide splice-site variant can change an mRNA (beta-globin examples).

1) Destroys a normal splice site and activates a cryptic one, giving an extended exon. 2) Destroys a splice site, causing exon skipping (e.g. exon 2 missing). 3) Creates a new splice site, causing an extra exon to be inserted.

What is alternative splicing?

Producing different mRNAs from one pre-mRNA by joining exons in different ways, so one gene can make several proteins. Proteome size is not directly related to gene number.

How common is alternative splicing?

Up to 95% of multi-exon genes, with an average of about 7 transcripts per gene (not all make functional protein).

Give an example of tissue-specific alternative splicing.

The alpha-tropomyosin gene is spliced differently in different types of muscle. Regulation can be negative (a regulator prevents splicing) or positive (a regulator promotes splicing).

What is the SLO gene example?

SLO encodes a Ca2+-regulated K+ channel. One splice variant in high-frequency hair cells has low calcium sensitivity; inclusion of the STREX exon in low-frequency hair cells gives high calcium sensitivity.

Why does alternative splicing matter for variant analysis?

A lab may miss a variant if it only assesses one transcript. In a 2019 epilepsy panel study (292 genes), 96% of genes were assessed on only one transcript, though 85% of genes have alternative transcripts and 30% have at least one alternative coding region.

Case: CDKL5 reanalysis. What was learned?

Trio exome in an infant with refractory spasms was non-diagnostic. Reanalysis found a variant that looked intronic in the canonical transcript (NM_003159.2) but was in exon 18 of NM_001323289.1, the most abundant CNS isoform, so loss of function made sense.

Case: OFD1 variant in a child with autism. What was learned?

A maternally inherited exon 10 variant in X-linked OFD1 did not fit (no orofacial-digital phenotype). gnomAD had several variants in the same exon and alternative splicing sometimes removes exon 10, so the variant was probably not causative.

What two complexes drive 3' end formation?

Cleavage stimulation factor (CstF) and cleavage and polyadenylation specificity factor (CPSF). They travel with RNAP II and transfer to the 3' signal sequences on the emerging pre-mRNA.

What are the key sequences for 3' end formation?

AAUAAA and the GU/U-rich element, specified in the genomic DNA.

Describe polyadenylation.

After cleavage, poly(A) polymerase (PAP) adds about 200 A residues using ATP, 5' to 3', without a template. Poly(A) binding protein (PAB) binds the emerging tail, determines its length, and stays bound during export and translation.

What does the mature 3' end do?

Protects the mRNA from degradation and helps target it to the cytoplasm for translation.

How can alternative 3' end use change a protein?

It can change the C-terminus. Example: the antibody switch. Membrane-bound antibodies have a C-terminal region embedded in the membrane; cleavage and polyadenylation at a different site removes it to make secreted antibodies.

How does eukaryotic transcription terminate?

RNAP II continues for several hundred nucleotides past the cleavage site. The downstream transcript lacks a cap and is rapidly degraded, and this degradation seems to signal RNAP II to stop.

Summarize mRNA processing in one line.

RNAP II makes pre-mRNA, which is quickly capped (meGpppG, 5'-5'), spliced (possibly alternatively), and cleaved and polyadenylated at the 3' end (possibly at an alternate site).

What is FSHD and how is it inherited?

Facioscapulohumeral muscular dystrophy. Autosomal dominant (about 10-30% de novo), prevalence about 1 in 38,000-80,000, with slowly progressive, often asymmetric weakness of face, scapular and upper arm muscles.

What is the typical FSHD clinical course?

Onset usually 15-30 years but variable; highly variable expressivity even within families (up to 30% of carriers non-penetrant or minimal); slow progression with possible periods of stability; about 20% need a wheelchair after age 50; normal lifespan

key characteristic of FSHD

scapular winging

Where is the FSHD locus and what gene is involved?

4q35. D4Z4 macrosatellite repeats contain the DUX4 gene (double homeobox 4), which is normally epigenetically repressed after development.

What is D4Z4 and how many repeats do affected vs unaffected people have?

A tandem array of 3.3 kb CpG-rich repeats, each containing a full DUX4 open reading frame. Unaffected people have 8-150 repeats; people with FSHD1 have 1-10 repeats (contraction). BUT not everyone with a contraction in this region has a phenotype!

Why is D4Z4 contraction necessary but not sufficient for FSHD?

Contraction loosens chromatin so DUX4 can be transcribed, but a similar contraction on chromosome 10 causes no symptoms, and not everyone with a 4q contraction is affected.

What second requirement does FSHD1 have besides contraction?

A permissive 4qA distal sequence variant (not 4qB). 4qA contains a cleavage/polyadenylation signal (ATTAAA) that makes the DUX4 transcript stable and allows DUX4 protein to be made.

What is FSHD2 and how does it differ from FSHD1?

About 5% of patients. They have 9-20 repeats (not in the type 1 range) plus mutations in SMCHD1 (about 85%) or rarely DNMT3B, which affect chromatin density. Same phenotype from a different gene (locus heterogeneity). FSHD1 is about 95%.

What is the disease mechanism in FSHD?

Toxic gain of function: DUX4, a transcription factor normally restricted to early embryo, is inappropriately expressed in adult muscle and triggers cytotoxic cascades and muscle cell apoptosis.

Which themes does FSHD tie together?

Active vs inactive chromatin, 3' end formation and mRNA stability, inappropriate transcription factor expression, and locus heterogeneity (DUX4 vs SMCHD1).

Why must mRNA be exported, and what is left behind?

Translation happens in the cytoplasm. Introns, 3' end fragments, unprocessed or aberrantly processed RNAs and other RNA types stay in the nucleus. Mature mRNAs are recognized by the nuclear pore complex.

What marks an mRNA as ready for export?

It remains associated with the cap binding complex, the exon junction complex, and poly(A) binding proteins. If splicing snRNPs are still present, the RNA is marked for splicing rather than transport.

What happens as mRNA is exported?

Proteins let the mRNA pass through the nuclear pore, and protein synthesis factors begin assembling as it exits.

example of combining alternative splicing and alternative 3’end formation

alpha-tropomyosin gene