Lecture 6: Regulation of the Transcriptome

review of rna processing and coupling transcription

• in eukaryotes, when does RNA processing occurs

• When does coupling occur?

  • How is it regulated

• RNA processing done before transcription is even complete

• During transcriptional elongation

  • CTD of RNA pol binds to RNA processing proteins and transfers to the RNA at the appropriate time to bind

  • Regulated by phosphorylation of RNA pol

    • Depending on the phosphorylation pattern; different proteins will bind

RNA transport out of the nucleus

• What specific RNA does it transport

  • How can it identify the specific type?

    • Two things it looks four

      • Three subtypes

• How many RNA is transported out vs how much the nucleus currently has in total

• In the nucleus: what happens if the specific RNA has not been formed

• transporting the mature rNA (with all modifications complete) out of the nucleus

• Markers of mature mRNA must be acquired for export

  • Cap binding complex (CPC) - found after G cap placed

  • Exon junction complexes (EJC) - found after splicing

  • poly-A-binding proteins - found after poly A tail placed

• Marker of immature mRNA must be lost before export

  • Proteins involved in RNA splicing (e.g. snRNPs)

• 1/20 RNA will leave the nucleus

• Any RNA not processed correctly into the mature mRNA will degrade in the nucleus by exosomes

Post-transcriptional Gene regulation: mRNA quality Control

• What are some issues once the mRNA are transported out of the nucleus

  • Why is this also an issue for the cell (one possible consequence)

• Some mRNAs are incompletely process or damaged in the cytosol once transported

• Prevent the production of weird, and unsuual proteins that could potentially be toxic to cells

Review of Translation

• tRNA match the amino acids to codons in the genetic code

• MRNA message is decoded in ribosomes made up of proteins and RNA molecules

  • Ribosomes: resource-intensive to make (made up of a lot of proteins

• Amino acids are added to the C-terminal end of the growing polypeptide chain (synthesizing proteins from 5’ to 3’ or N to c-terminus)

mRNA Quality control in eukaryotes

• Eukaryoteic intuition factors binds to the mRNA (eIFs) - two kinds

  • EIF4E binds to the 5’ cap

  • EIF4E binds to the poly-A binding protein

• Once the mRNA loops

• Recruit small ribosomal complex which will initiate translation at first AUG downstream of 5’ cap (some exceptions)

• Once they find the sequence; the large ribosomal subunit will bind to the small ribosomal complex

• Ensures that both ends of mRNA are intact

  • A loop is a mRNA quality control; indicating that the

  • This entire process occurs in the cytosol

• (EJC) also stimulates translation ensuring proper

  splicing

EJC helps with non-sense-mediated mRNA deday

• what is it’s main job

• Recall: EJC are where the intron use to be before they were spliced

• prominent mRNA surveillance system

• surveys for nonsense (STOP) codons in the “wrong place”

• Indicates improper splicing

  • If there is a premature stop codon: its stops and the 2nd EJC does not get removed, and upf proteins will trigger the degradation the mRNA

EJC helps with non-sense-mediated mRNA deday - normal splicing

• still requires the loop and eIFs, even if not shown on the diagram

• Steps

  • The ribosome binds mRNA as it emerges from the nuclear pore

  • EJCs are displaced by the moving ribosome

  • The stop codon is in the last exon

  • No EJCs remain bound when the ribosome reaches the stop codon

  • mRNA is released in the cytosol

    • the first ribosome still makes protein

EJC helps with non-sense-mediated mRNA deday - abnormal splicing

• The ribosome binds mRNA as it emerges from the nuclear pore

• EJCs are displaced by the moving ribosome

• The stop codon is premature

• The EJCs remain on the mRNA when the ribosome reaches the stop codon

• mRNA is degraded (mediated by Upf proteins)

**if there is no EJCs by the stip it meet the premature stop codon, the EJC cannot mediate the degradation (there is no EJC to actually help)

The importance of nonsense-mediated mRNA decay

• May have played an important role in the evolution of eukaryotes by allowing the selection of DNA rearrangements or alternative splicing patterns that produce full-length proteins

• Important role in cells of the immune systems where extensive DNA rearrangements occur to produce antibodies

• Also plays a role in many human disease cause by mutations that produce aberrant proteins. Cells can degrade aberrant mRNA and allow functional protein to accumulate

• occur in the cytosol

• Post-transcriptional gene regulation

MRNA quality control in prokaryotes

• Ribosomes stall on broken or incomplete mRNAs and do not release

• A special RNA tmRNA is recruited to the A site

• Carries an alanine amino acid

• Acts as both tRNA and mRNA

• Broken mRNA is released

• Alanine is added onto the polypeptide from the tmRNA, which acts like a tRNA but with no anticodon-codon binding

• The ribosome translates 10 codons from the tmRNA, which now acts as an mRNA

• The 11 amino acid tag is recognized by proteases that degrade the entire protein

  • 11th codon is the stop codon

Kind of degradation in prokaryotes and eukaryotes

• method of degradation of both of them

The ribosome translates 10 codons from the tmRNA, which now acts as an mRNA

The 11 amino acid tag is recognized by proteases that degrade the entire protein

Processes carried out by an exonuclease (deadenylase) when mRNA reaches the cytoplasm – acts as a timer of mRNA lifetime

• eats the poly-A tail, until reaches 25 nucleotides of A-tail

Two possible things that happens next

• Breaking of 5’ cap

• Continue degradation and losing all A tail

e.g. of mRNA stability and Transferrin

Transferring receptor

• imports iron into the cell

• Low iron: receptor is express

• High iron: no receptor is expressed

Low iron

• mRNA stabilized by cytosolic aconitase that binds 3’UTR

High iron

• aconitase binds iron and undergoes a conformational change

• mRNA released

• exposes 3’UTR endonucleolytic cleavage site (polyA removed); mRNA is degraded

mRNA stability and Deadenylase

Another example of protein-regulated mRNA stability…

There is competition between mRNA translation and mRNA degradation

➢ deadenylase that shortens the poly-A tail binds the 5’ cap (like eIFs)BIO230 Lecture 1-6 21

MiRNAs

non-coding RNAs called microRNAs (miRNAs) also regulate mRNA stability

(>2000 in humans)

• miRNAs base-pair with specific mRNAs

• synthesized by RNA polymerase II and get a 5’ cap and poly-A tail

• after special processing, the miRNA associates with a protein complex called an RNA-induced silencing complex (RISC

• enzymes does cropping

• Moves into cytosol

• dicing done by the diver enzyme (the diver cuts long double stranded RNA)

RISC seeks mRNA with complementary nucleotide sequences

• A protein of RISC called Argonaute plays a critical role in base-pairing miRNA with mRNA

• Two possible outcomes are possible

For the less extensive math:

• blocks translation

• can also be reused

RNA Interference (RNAi)

➢ Double-stranded RNAs that end up suppressing the gene expression of other RNAs in a sequence-specific manner

➢ The proteins used in the miRNA regulatory mechanisms also serve as a defense mechanism against foreign RNA molecules

➢ Found in eukaryotes, including fungi, plants, and worms

SiRNAs

• Many viruses (and transposable elements) produce double-stranded

RNA as part of their life cycles

• RNAi destroys double stranded RNA

• Initiated by Dicer protein complex that cuts the RNA to form siRNAs

• siRNAs can interact with Argonaute and RISC proteins and follow the miRNA route to destroy double-stranded RNA or another method (RNA degradation)

Other method (DNA silencing)

• siRNAs can also regulate transcription

• siRNAs interact with Argonaute and the RNA-induced transcriptional silencing (RITS) complex

• Interacts with newly transcribed RNA

• Recruits chromatin modifying enzymes to silence the DNA (no more transcription at all from the DNA itself)

CRISPR-Cas Prokaryotic Immunity

• not a mechanism found in eukaryotes (however, you can put the CAs protein into eukaryotic cells and small RNAs to cut things inside the eukaryotic cells)

• 100% prokaryotes

• short fragments of viral DNA integrate into the CRISPR region of the genome and become templates to produce crRNAs (CRISPR RNAs)

• Aside: The viral genes are transcribes into pre-crRNA and processed using CAs protein to create crRNAs (targeting mechanism) that can cleave a previous known viral DNA

• Viral DNAs complementary to CRISPR regions are directed for degradation by Cas (CRISPR-associated) proteins

• Similarly to (Eukaryotic): use of small single-stranded RNA

Differences

• Argonaute is cutting RNA

• The cas proteins cut DNA