Lecture 14: Control of Gene Expression Part 2 - Regulation of mRNA Levels & Post-Transcriptional Regulation Flashcards
1
Q
Post-transcriptional Regulation
A
- Transcriptional regulation is not the only level at which gene expression is regulated
- Growing realization that most eukaryotic genes are regulated by alternative splicing
- > A gene with even a few exons can produce many different mRNAs via alternate splicing - “splice variants”
- > Some variants may simply lack one or more exons
- > Sometimes there are mutually exclusive alternate exons
- > May be regulated by splicing repressors or splicing activators
- Similar theme: alternative poly-A site addition
- mRNA can also be regulated after processing is complete
2
Q
More post-transcriptional regulation
A
- HIV uses regulated nuclear export to allow RNA molecules containing some introns to be expressed from the nucleus - important for HIV life cycle
- Cells can use regulated cytosolic localization to place specific mRNAs at specific locations in the cell
- > Allows mRNA, and the encoded protein, to be concentrated in a particular part of the cell
- > Also allows inactive mRNA
- Some genes are regulated by RNA stability - mRNA is rapidly degraded under certain conditions
- > Shuts off expression of original gene
3
Q
Transferrin regulation
A
- In iron starvation, the cell must be able to import more iron and make sure the iron gets used properly
- > Iron in the bloodstream is attached to a molecule called transferrin
- > Transferrin receptor imports iron into the cell
- When there’s excess iron, we want to shut down iron import
- > Cytosolic aconitase unbinds the mRNA to attach to iron which reveals a endonucleolytic cleavage site
- > The mRNA meant to express transferrin receptors then gets cleaved and is no longer functional
4
Q
MicroRNAs (miRNAs)
A
- Are derived from precursors that fold into “hairpin” stem-loop structures
- After processing, a short double-stranded RNA is generated and associates with a set of proteins to form an RNA-induced silencing complex (RISC)
- One strand of the RNA is degraded, and the other makes base-pairing contacts with an mRNA target
- Depending on the degree of base-pairing, the target mRNA may be degraded, or translation may be inhibited
5
Q
Small inhibitory RNAs (siRNAs)
A
- Mediate the process of RNA interference
- Double-stranded RNA (dsRNA) is formed by base-pairing between the complementary regions of separate RNA strands
- dsRNA is cleaved by the Dicer nuclease to form short double-stranded RNAs: siRNAs
- As with miRNA, siRNA associates with proteins to form RISC, and target mRNAs are cleaved
- > Can go on to cleave much more mRNA than miRNA
- siRNAs can associate with a slightly different set of proteins to form an RNA-induced transcriptional silencing (RITS) complex, which inhibits gene transcription by modifying chromatin structure
- The siRNA pathway is thought to be an ancient antiviral defense mechanism (viruses often produce dsRNA, but eukaryotic cells rarely do)
6
Q
Regulation of Protein Translation
A
- Once an mRNA has been synthesized, protein amounts can still be regulated at the level of translation
- Information in the 5’ and 3’ untranslated regions (UTRs) can regulate translation efficiently as well as mRNA
- > Information to turn on or turn off translation
- > 5’ UTR RNA structure can allow binding of translation receptor protein that blocks ribosome access
- > RNA structure itself (e.g. hairpin) may inhibit ribosome scanning
- > “Riboswitch” structure may use binding of an ion or small molecule to switch between translation “on” and “off” states
- > Repressors binding to 3’ UTR can prevent communication between 5’ and 3’ ends of mRNA (required for efficient translation initiation)
7
Q
More regulation of protein translation
A
- In excess iron, ferritin stores away the iron and cytosolic aconitase binds to iron which unblocks translation of ferritin protein
- In iron starvation, cytosolic aconitase binds to ferritin mRNA so it isn’t made and iron isn’t stored
- Binding of translation repressor to 3’ UTR can block interaction of 5’ and 3’ mRNA ends
8
Q
Initiation factor eIF2
A
- Phosphorylation of initiation factor eIF2 can inhibit global protein synthesis
- > eIF2 uses GTPase motif to mediate binding of initiator met-tRNA to a small ribosomal subunit
- > eIF2B is a GEF (guanine nucleotide exchange factor) that catalyzes exchange of GDP to GTP, activating eIF2
- > Phosphorylation of eIF2 turns it into an inhibitor of eIF2B, blocking translation initiation
9
Q
Upstream open reading frames (uORFs)
A
- Context surrounding AUG can allow regulation by “upstream open reading frames” (uORFs)
- > More than one possible open reading frame
- > Open reading frame is a sequence starting with an AUG and ending with a stop codon, theoretically able to encode a polypeptide
- > These have more context, so ribosome can more easily recognize it
- > Some genes have short ORFs upstream of the “main” coding sequence - if the ribosome begins to translate a uORF, it will terminate with the stop codon and fall off the mRNA before reaching the main coding sequence
- > These normally just get read through
- > Phosphorylation of eIF2 turns decreases global translation initiation, allowing some ribosomes to “read through” uORFs to reach the main coding sequence - a strategy to selectively increase a few proteins during stress conditions (e.g. amino acid starvation)
10
Q
uORFs regulate translation of ATF4
A
- ATF4 is a transcription factor in responses to various stresses, including amino acid starvation
- Under non-stress condition, ATF4 translation is inhibited by uORFs
- Under stress condition, eIF2 is phosphorylated, reducing initiation at uORFs
- Some ribosomes can read through and initiate translation of ATF4
11
Q
Internal ribosome entry site (IREs)
A
- Allows ribosome to skip the first AUG by binding to an internal site
- This allows 2 different protein sequences to be derived from a single mRNA
- > Different initiation sites may lead to skipping of a signal sequence (required for secreted/transmembrane proteins), and so switching between cytosolic and secreted forms of a protein
- > IREs may sit between 2 separate ORFs, allowing independent simultaneous translation of 2 completely different proteins from one mRNA
- > Viruses use IRE-initiated translation to synthesize viral proteins while initiating host cell cap dependent translation
12
Q
Regulation of Protein Activity
A
- Protein function is heavily regulated by post-translational modifications
- Phosphorylation may directly affect protein activity, or may generate new binding sites for protein - protein interactions
- Various post-translational modifications can direct proteins to new cellular sites
13
Q
Regulation of Protein Stability
A
- Protein turnover is another point of regulation
- > How long a protein molecule lasts in a cell once it’s been synthesized
- Some proteins are highly stable (actin and tubulin), but some are rapidly degraded, and then resynthesized when needed
- Damaged or misfolded proteins must be destroyed to prevent accumulation of malfunctioning proteins
- The ubiquitin/proteasome system allows for regulated destruction of proteins
- > Targeted protein is polyubiquitinated
- > Proteasome recognizes polyubiquitinated protein and degrades it into short peptides
14
Q
The Proteasome
A
- Multi-protease complex
- Contains many proteases that can recognize many amino acids so a protein can be cut in many different ways
- At both ends of the molecule, there are ATP-dependent “unfoldases” that allow a protein to be threaded into the proteolytic core as an unfolded peptide chain
15
Q
More regulation of protein stability
A
- Proteasomal degradation is very energetically expensive
- > Must synthesize multiple copies of ubiquitin
- > Cutting of unfolded protein doesn’t require ATP
- > Ubiquitin ligation uses 2 ATP equivalents (ATP —> AMP) for each added ubiquitin
- > Unfoldedases require ATP to feed protein into proteolytic cylinder
- Other major cellular proteolytic organelle, lysosome, degrades proteins with no ATP requirement, but is non-selective
- Highlights the importance of having a mechanism for regulated protein degradation - cell is “willing” to spend lots of energy
