lecture 1: the dark side of the genome

RNAPI: rRNA (45% of activity) - the process

a 35s precursor transcript is cleaved into smaller intermediate sizes (32s → 20s + 27s → 18s + 5.8s + 25s)

RNAPII: mRNA (45% of activity) - the process

the starting transcript is pre-mRNA, which contains exons and introns (85%). the introns are looped out and removed, so the exons are linked together

RNAPIII: tRNA (10% of activity) - the process

begins as a precursor containing extra sequences on its ends. ribonucleases trims these off of the 5’ and 3’ ends

how much of a larger fraction is pre-mRNA degraded compared to pre-rRNA and pre-tRNA?

95%

what type of RNA does electrophoresis agarose gels show?

large RNA (rRNAs)

what type of RNA does electrophoresis acrylamide gels show?

small RNAs (tRNAs and other small stable RNAs (except mRNA)

why can mRNA not be seen via ethidium staining (on the gel)?

because it is unstable and heterogenous

1000s of varying mRNA lengths being expressed can be seen as a smear at the bottom of the gel

how does a pulse-chase experiment work?

• a radioactive ‘pulse’ of uridine is added to the growth medium to label newly synthesised RNA

• a non-radioactive ‘chase’ of uridine is added to track where RNA goes over time (separate experiments are prepared for nuclear and cytoplasmic extracts)

• the extracted RNA is analysed via ultracentrifugation

how is hnRNA distinguished from rRNA?

rRNA has a much higher GC content

heterogenous nuclear RNA (hnRNA)

all the other RNA molecules including mRNA

how much of the nonribosomal (hn)RNA survives long enough to be transported out of the nucleus?

2-5%

pervasive transcription

the whole genome is transcribed (not just the protein-coding regions)

how many long noncoding transcripts do human cells produce?

~36000

many noncoding transcrits are never translated or exported, instead they are…

rapidly degraded in the nucleus

what are some of the benefits of pervasive transcription?

• molecular interactions → transcripts can act like a scaffold for various proteins

• RNA sponges

• shapes/ modifies local chromatin organisation and packaging

• assists in recruitment or eviction of transcription factors

• transcription interference

RNA sponges

noncoding RNAs can prevent the small RNAs from interacting with the target with target mRNAs to stop them from being silenced or degraded

transcription interference

active transcription in 1 region can physically block or interfere with the transcription of neighbouring genes

intergenic regions

noncoding regions between between genes give rise to long intergenic RNAs (lncRNAs), enhancer RNAs (eRNAs) and transposon elements

RNAPII promoters are bidirectional, meaning…

it doesn’t need to just go forward to make mRNA, by going in the opposite direction, it can generate various noncoding transcripts

what are some of the repressive mechanisms that the cell has?

• ssu72: gene looping + modifies the C-terminal of RNAPII to promote early termination

• rpd3: causes histone deacetylation, closing the chromatin structure

• rrp6: an exoribonuclease that hunts down noncoding transcripts

under normal conditions, how much of new tRNAs are degraded as apart of quality control (degrading incorrectly processed/assembled)?

~50%

what happens when ribonucleases are mutated?

the cell accumulates high levels of tRNAs

what processes are subjected to quality control?

mRNA synthesis, tRNA maturation and ribosome biogenesis

why does human biology rely on pervasive transcription?

• it acts as an evolutionary capacitor

• can experiment without lethal consequences (can produce advantageous or disadvantageous mutations)

how can the enzymes for degradation differentiate between correct and aberrant RNAs?

• 5’ cap

• 5’ phosphate group

• 3’ poly-A tail

what can histone mRNAs like MALAT form at the 3’ end?

a triple helix/ hairpin shape

what kind of diseases can cellular stresses via unwanted RNAs in the nucleus cause?

neurological diseases like motor neuron disease and autoimmune diseases

ALS

accumulation of toxic RNA repeats that ribonucleases cannot breakdown causing them to clump together that can trap proteins