Lecture 22: Post-Transcriptional Control

Eukaryotic ribosome activity is

extensively regulated

Insulin receptor allows

Cells to respond to insulin release into the bloodstream

When activated, the insulin receptor causes

Cells to take up glucose for ATP production

Turning off a gene at many points

Control at 3 levels: 1. Transcriptional control: turning off the gene 2. Translational control: degrading the transcripts, preventing translation 3. Post-translational control: actually degrading the protein itself

Translation initiation

In eukaryotes, translation initiation requires the 5' cap 1. The small subunit of the ribosome assembles at the 5' cap and scans for AUG (start codon) 2. At the AUG, the large subunit assembles in translation begins

What makes this miRNA specific to the gene?

It's going to have a complementary base sequence to part the gene

Exons

Joined together

Complex assembly

Many proteins only activate as part of larger complexes -Ex: photosystems in photosynthesis

Regulation occurs through

mRNA sequence and mRNA binding proteins

miRNA pathway

miRNA transcript exported from nucleus to cytoplasm -5' cap and poly A tail are removed -miRNAs have regions of self-complementarity, leading to the formation of dsRNA and a hairpin loop -specific enzymes recognize dsRNA and cleave the miRNA, releasing one strand -miRNA assembled in the RISC complex (RNA induced silencing complex)

Degrading the mRNA stops protein production, but what about the existing protein?

One way to turn off a protein gene product is to degrade the protein via the proteasome

In many cases, the lifespan of an mRNA is controlled by

Proteins binding to the 3' UTR of mRNAs and the length of the poly A-tail

Chemical modification

Proteins can be activated (kinase) or deactivated (phosphatase) by covalent modification, such as the addition of a phosphate group. The modification changes the confirmation of the protein -Ex: phosphorylation/dephosphorylation

Post-translational control is a huge world regulating

Proteins localization, activity, and stability -Ex: folding, degradation, chemical modification, and transport

Targeted distraction of proteins

Proteins that have covalently attached ubiquitin are recognized by the proteasome and degraded

Regulation of alternative splicing is controlled by

RNA-binding proteins specifically expressed in muscle cells that blocks the spliceosome recognition of exon 11

miRNA biogenesis

Slide 22 — transcription —> RNA processing —> export to cytoplasm —> further processing (instead of moving to translation) — much smaller sequence than regular protein-coding gene, about 13 nucleotides

Example of mRNA stability

Slides 18 and 19

Intron

Spliced out

Why do cells use miRNAs? Why not just stop transcribing the gene?

Stopping transcription leaves plenty of mRNA still in the cytoplasm to be translated. If you want to turn off aging completely, you need to both stop transcribing it and degrade the mRNA and turn off/degrade the protein -see slide 25 and 26

5' cap modificaiton

once 5' to 5' phosphate linkage is formed, The mRNA does not have a free 5' end which typically are targeted for a degradation, making the mRNA more stable and necessary for translation

miRNA-RISC complex

Transcriptional inhibitor 1. Find target mRNA 2. Face-pairing between mRNA and RISC miRNA —1. Inhibit translation —2. Degrade Target mRNA

Protein localization

Transport of some proteins occurs automatically, for others proteins transport is highly regulated — Signal localization: ability and inability to transport

snRNPs

(small nuclear ribonucleoproteins) composed of RNA and protein molecules, recognize the splice sites, join with additional proteins to form a spliceseome —are catalysts = ribozymes

Estrogen receptor regulated localization

-The estrogen receptor (ER) is a regulatory transcription factor -The ER is normally in the cytoplasm, where it is in active and cannot bind to DNA 1. When estrogen gets inside the cell, it binds to the ER 2. The ER then moves into the nucleus, binds enhancers, drives transcription (very tightly regulated post-transcriptional control)

mRNA stability

-The stability of mRNAs in the cytoplasm is highly variable. Some mRNAs are degraded rapidly, allowing for only a short period of translation, while others are quite stable

Regulation of mRNA processing

-The world of mRNA control is a rapidly involving field -we are learning mRNA control is incredibly complex and a very important aspect of gene regulation -The human genome contains over 1500 mRNA-binding proteins

MRNA processing events in eukaryotes

-addition of "cap" to 5' end of RNA -addition of poly A "tail" to 3' end of RNA —— these modifications stabilize the RNA and help to promote translation

RNA processing events in eukaryotes

-intron: portion of primary transcript removed from the mature mRNA (processed transcript, do not contain coding sequences) -Exon: portion of primary transcript that is retained in mature mRNA (exons contain both coding sequences and UTR sequences) -UTR: untranslated region. The portion of the mRNA outside of the coding sequence

Alternative spicing with liver cells versus muscle cells

-liver cells do not express this mRNA-binding protein which leads to a receptor with low insulin affinity (exon 11 present) —— in the liver, 36 nucleotides constituting Exon 11 are included in the messenger RNA) -muscle cells express an RNA-binding proteins in which blocks exon 11 inclusion leading to a receptor with high insulin affinity —— in skeletal muscle, alternative splicing excises exon 11 along with the flanking introns

Chaperones

-many proteins require chaperones as a normal part of synthesis/folding -chaperones can bind to newly synthesize proteins as they exit the ribosome on or even provide a full environment when the protein can fold -prevents hydrophobic aggregation

Post-transcriptional gene expression control summary

-once a pre-mRNA is made, a series of events must occur if the gene product is going to affect the cell. Each event offers a opportunity to regulate gene expression -post-transcriptional regulation is anything that occurs after transcription -everything from mRNA processing, to mRNA stability to translation and protein activation can and often is tightly regulated

Constitutive protein localization

-proteins function in a specific subcellular location -for most proteins, localization is to a specific place (Ex: DNA polymerase needs to stay in the nucleus) and happens constituently: as soon as the protein is made, it is localized to its place of function which is highly regulated

Regulated processes

-ribosome small sub unit assembly at 5' cap -ribosome scanning -ribosome translation elongation on long the transcript -ribosome pausing

RNA interference

-small double-stranded mRNA is called microRNAs (miRNAs) interfere with mRNAs

Influence stability of the mRNA

1. Increase the stability/shelf life of mRNAs, being able to be translated for a longer period of time 2. Decrease the stability of mRNAs How? These mRNA binding proteins can recruit RNases or protect the transcript from RNases

Proteasome (anti-chaperone) degrades ubiquitin-tagged proteins by

1. Targets misfolded proteins = cleaning up non-functional proteins 2. Controls protein lifespan = regulation of gene expression by regulating protein amount

Humans make over

2,500 different miRNAs

Splicesome

A larger assembly of proteins and snRNA

Other cells especially liver cells which store glucose as glycogen need

A low affinity insulin receptor

90% of human genes have

Alternative transcripts, making humans as complex as they are

Translation is highly regulated because

By the sequences in the mRNA, especially in the 5' and 3' UTRs -proteins can bind to sequences in the 5' UTR to transport the mRNA to particular locations in the cell, or can affect translation initiation directly -translation is often regulated by sequences in the 3' untranslated region, sometimes by binding with specific proteins

miRNAs can

Degrade mRNAs or block translation of mRNAs

miRNAs target specific RNAs by

Direct base-pairing

5' UTR

From 5' cap to AUG (start codon) -Reading mRNA, won't translate until start codon

Both high affinity and low affinity insulin receptors are synthesized

From the same gene, mRNA is just spliced differently

miRNAs are encoded by

Genes, but never translated

Muscle cells need a

Highly affinity insulin receptor because they have such a high demand for glucose

Alternative splicing

Produces more than one mature mRNA and proteins from the same gene

poly A tail

The longer the poly A tail, the more stable the mRNA which allows the mRNA to leave the nucleus for translation

The insulin receptor gene is expressed in many genes, including liver and muscle cells however

There are two different protein isoforms of the IR protein that are produced, with high affinity and low affinity

mRNAs are degraded by enzymes called ____________.

ribonucleases (RNases)

RNA-binding proteins can control

which sequences are spliced out and which are retained