20. RNA processing and Control of gene expression

Transcription initiation

• direct infulence of A and R on initiation

• activators, repressors, remodeling proteins and chromatin structure

• methylation

• hormones and other external signals

RNA processing

• alternative splicing and trans-splicing

• polyadenylation

posttranscriptional / pretranslational control

• localization of mRNA

• RNA editing

• POst-transcriptional silencing by siRNA or miRNA

• translational control switch

• RNA stability (degradation and stabilization)

translational control (mRNA/ribosomes)

• phosphorylation of translation initation factors

• upstream AUG codons

• IRES

protein activity control

= posttranslational modification and transport

simple transcription units

constitutive splicing

complex transcription units (at least 5% of eu)

alternative splicing (regulated by specific splicing factors)

expression of complex transcription units controlled

• regulation of splciing cleavage-polyadenylation

• alternative processing

alternative processing of complex pre-mRNAs regulated according to

• cell type, developmental stage or change in environmental conditions

regulating sequences on pre-mRNA

exonic (or intronic) splicing enahncers, ESE (or ISE) or silencers, ESS (or ISS)

trans factors

proteins (splicing activators and repressors) regulate splicing thru binding to splicing enhancers and silencers, respectively on pre-mRNA

splicing activators

have RNA-binding motif as well as Arg/Ser rich motif (SR proteins) for attracting splicing machinery

splicing repressors bind RNA but do not have RS domains

→ repressors cannot recruit the splicing machinery

they block the use of the specific splice sites by bidning them

tissue specific alternative splicing

fibronectin gene exons are spliced in different ways to produce two isoforms of the protein.

• one in hepatocytes (2 fewer exons) and the other in fibroblasts - constitutive alternative splicing

stable tissue specific pattern

developmental

Dscam plays a widespread role in regulating formation of neuronal connections in Drosophila. different neurons express different combinations of Dscam isoforms endowing each neuron with a discrete moelcular identity. 95 “variable” exons out of 115 total in 4 different blocks (one exon/block for 38,106 different isoforms)

sex determination alternative splicing in drosophila

regulated splicing of alternative exons results in differential expression in male and female dorsophila embryos (cascade effect) → role in sex determination

different C-terminus in the synthesized proteins could lead to

tissue specific alternative polyadenylation

alternative polyadenylation - common mechanism in all eukaryotes

• 51% of human genes undergo

• modulate gene expression in many eu

• implicated in different disease

U1A protein from U-snRNP (small nuclear ribonucleoproteins)

• bind to two identical sites upstream of the poly a singal in it OWN pre-mRNA - autoregulation

• binding will inhibit ONLY polyadenylation

• pre-mRNA for U1A is degraded

  • when excess U1A present

trans-splicing found mostly in protozoa

mRNAs are constructed by splciing of separate RNA molecules, spliceosome joins exon of one gene with the exon of another gene

discontinous goroup II trans-splicing

plant organelle genes spliced together in complex processes (cis and trans splicing)

only functional mRNAs are exported

5’ cap is recognized by transport machinery

in nucleus the pre-mRNAs do not exist as free molecules

associated with proteins hnRNPs

proteins associated with processed mRNA - nuclear mRNP (messenger RNPs)

are exported together with mRNA into cytoplasm

as mRNA-nuclear mRNPs pass through nuclear pore, nuclear mRNPs get replaced with

cytoplasmic mRNPs

pre translationional control: localization of mRNA to specific regions

• transport of mRNA close to teh sites where the protein product is required

• requires different elements that allow diff mRNAs to be recognized as targets for transport: elements mapped within 3’UTR

proposed model of ZBP1-mRNA localization

1. ZBP1 shuttles into and out of the nucleus

2. if it encounters the nascent zipcode seqeunces, it binds there as the nascent RNA finishes transcription and processing/polyadenylation

3. once in cytoplasm, it associates with cytoskeletal elemetns and localizes to leading edge

4. once detached from the mRNA, ZBP-1 returns to the nucleus

RNA sequence is altered in a

single-nucleotide, site-specific manner

RNA editing in mammals

1. deamination of A to I (inosine)

2. deamination of C to U

RNA editing in mitochondria of protozoa, mitochondria and cholorplasts in plants

addition or deletion of U residues - guide RNAs (gRNA)

U-insertion

1. anchor region of gRNA: bidning of gRNA to mRNA due to high complementarity between sequences

2. editing region of gRNA: partial complementarity with mRNA guides insertion of Us

RNA interference

ability of small double stranded or single stranded RNA molecules to induce sequence specific gene splicing through bidning

post transcriptional gene silencing (PTGs) by small RNA molecules

1. specific protein complexes - DICEr will cut noncoding imperfect dsRNA hairpins → miRNAand/or dsRNAs that are typically hundreds of base pairs → small interfering RNAs

2. siRNA and miRNAs get incroporated as a single-stranded RNA into nuclease complex - RNA-induced silencing complex (RISC)

3. RISC recognizes complementary mRNA and depending on the % of complementarity with the target mRNA it will stop translation, cleave mRNA or induce degradation

miRNA - natural antisense RNAs

work as regulatory factors in development

Gerritin mRNA

stores excess iron inside cells

• has IRE in its 5’ UTR → regulation is at the level of translation

Transferrin receptor mRNA

imports iron into cells

• has IRE in its 3’ UTR → regulation is at t he level of mRNA degradation

an iron-responsive bidning protein (IRP or IRE-BP) binds to the IRE(s)

IRE-BP is sensitive to iron concentration

when Fe is low in the cell

1. IRE-BP is active: binds to the IRE in both mRNA’s UTRs

2. ferritin mRNA translation stopped - ferritin synthesis prevented

3. transferrin receptor mRNA degradation is prevented, transferrin receptor is translated

when Fe is high in the cell

1. IRE-BP binds Fe changes conformation, becomes inactive and cannot stay bound to IRE

2. ferritin mRNA is unblocked, storage protein is synthesized and Fe gets stored

3. transferrin receptor mRNA is degraded, transferrin receptor is not synthesized ( no longer needed as fe levels rise)

what determines mRNA longevity (stability)

• Stabilizing elements: Found in the 3′ UTR and coding sequence; increase mRNA lifespan.

• Instability elements: Often in 3′ UTR, signal rapid degradation (e.g., AU-rich elements, ARE = "AUUUA").

• ARE Binding Proteins (ARE-BPs):

  • Some promote stabilization.

  • Others promote degradation (can act from both 3′ and 5′ ends).

  • Also influence ribosome accessibility to mRNA.

• Degradation pathways:

  1. Deadenylation → decapping → 5′→3′ degradation.

  2. Deadenylation → exosome recruitment → 3′→5′ degradation.

• The balance between mRNA degradation and synthesis controls the level of specific mRNAs in cells.

1. 3’ UTR regulation in cancer cells

1. genetic alterations in cis elements

2. altered elvels of trans-acting factors

   • increase of ARE-BPs

   • decrease of miRNAs levels