Paul Gardner

Question 1

outline the importance of ncRNAs?

Answer 1

ncRNAs are central to life’s major processes; some studies suggest >80% of human genome encodes ncRNAs

ncRNAs involved in central dogma reactions:

Y RNA important for replication initiation

spliceosome packed with ncRNAs; potentially involved in catalaysis of splicing reaction

RNase MRP made up of RNA; chops up long transcripts to produce ribosome

snoRNP guides modifications of rRNAs

RNAseP processes precursor tRNAs

alot lot of these prob ribozymes

Question 2

what is a vault organelle?

Answer 2

example of ncRNA

eukaryotic organalle; function not fully understood yet

larger than ribosome and highly conserved across much of life

knockouts are viable

Question 3

what is a PrfA thermoswitch?

Answer 3

example of ncRNA based mecahnism of thermoreg of translation

PrfA protein a master coordinator of a major virulence regulon in Listeria; translation of PrfA regulated by thermoswitch upstream of start codon

cold temps - forms secondary structure preventing ribosomal binding = no translation; ingest listeria our bodies heat up RNA to about 37 degs; RNA structural change and ribosome can bind

Question 4

ncRNAs are some of the most ___ molecules of life?

Answer 4

highly conserved

Question 5

what are self-splicing RNAs?

Answer 5

RNAs that when transcribed fold into secondary structure and cleave themselves out of RNA

Question 6

how was it identified that transcription is pervasive?

Answer 6

early evidence from EST and microarrays suggested pervasive transcription

later confirmed by ENCODE and RNA-seq experiments

Question 7

how do we define ncRNAs?

Answer 7

can be defined in many ways; two extremes:

All RNAs other than mRNA i.e. transcription alone sufficient to define a ncRNA

RNA genes which produce transcripts functioning as structural, catalytic or regulatory RNAs, other than encoding a protein i.e. requires evidence of function; function often also used in ambiguous way (e.g. causal vs selected effects)

Question 8

what is the evidence suggesting a lot of transcription is noise?

Answer 8

some studies have shown a lot of transcription occurs from random DNA sequence

TF binding sites cover the genome

we have a big genome which can be broken down into functional and junk bits; both can end up getting transcribed

Question 9

discuss the proportions of cellular mammalian RNAs by mass and by molecules?

Answer 9

RNA by mass; rRNA makes up 80-90% due to large size

RNA by number of molecules; tRNA makes up most

so cellular RNA pool very much dominated by rRNAs and tRNAs

Question 10

why is it important to consider the makeup of cellular RNA pool when using RNA-seq methods and what strategies are used as a result?

Answer 10

RNA-seq can be used to sequence pool of transcripts in cell BUT if cellular RNA pool dominated by rRNAs and tRNAs the sequences will be dominated by these

can size select into long and short RNA fractions; can deplete rRNA to better find unique transcripts like ncRNAs; can enrich mRNAs using polyA enrichment to better quantify protein-coding transcripts

Question 11

what are catalytic RNAs?

Answer 11

RNA that catalyse reactions aka ribozymes

Question 12

outline how RNA can acts as an enzyme?

Answer 12

RNA can store genetic information and catalyse reactions e.g. RNA viruses

discovered in early 1980s RNA can act as an enzyme (ribozymes) e.g. self-splicing introns

this was identified to be a two-step reaction called transesterification involving cleavage and ligation

Question 13

what is transesterification?

Answer 13

exchanging organic functional R group with organic group of an alcohol

the two-step reaction used by ribozymes to catalyse biochemical reactions

Question 14

outline the RNA world hypothesis?

Answer 14

solution for question of which came first; genetic encoding info in DNA or enzymatic activities of protein

possible neither; RNA may have been progenitor of modern DNA/RNA/protein-based life

Question 15

what is the ribozyme RNase P?

Answer 15

is a ribonucleoprotein (RNP) found in all branches of life

required for maturing precursor tRNAs; reaction catalysed by ncRNA component

Question 16

how is the ribosome a protein synthesis ribozyme?

Answer 16

eukaryotic and bacterial ribosome both large complexes composed of RNA. protein and have large (50s) and small subunit (30S); a lot of similarity when aligned

used to be thought protein in ribosome drive translation but they not that conserved and located at periphery of complex; catalytic core of ribosome highly conserved across all life and entirely RNA

Question 17

discuss the ribosome catalytic core?

Answer 17

aka peptidyl transferase centre; important location for translation reactions i.e. taking charged tRNAs w aa attached, chopping them off, adding to peptide chain

key interactions include with mRNA (RBS w small subunit) and tRNA interactions w large subunit via CCA tail

major target for antibiotic development (and just the ribosome in general)

Question 18

what is the spliceosome?

Answer 18

takes pre-mRNA and chops out introns between all the exons to produce mature mRNA w no introns

does this by assembling diff RNAs and proteins on mRNA; RNAs do most of the key interactions on complementary regions of mRNA

so spliceosome forms complex on mRNA and catalyses transesterification (cleavage + ligation)

Question 19

what evidence is there to suggest the splicesome might be a ribozyme?

Answer 19

catalytic core of spliceosome comprised of RNA and it shares a lot of similarities (structure, reactions) with self splicing introns

Question 20

outline splicesome assembly and splicing reactions?

Answer 20

involves multiple stages, transient interactions between several ncRNAs, mRNA and proteins

two-key chemical steps:

• cleavage of 5’ exon-intron boundary
• ligation of 5’ exon w 3’ splice site

i.e. two transesterification reactions

Question 21

what are the key interactions of the spliceosome?

Answer 21

between splice sites, branch site and spliceosomal RNAs

Question 22

discuss the catalytic core of the spliceosome?

Answer 22

U2 and U6 snRNAs create active centre similar to that of group II splicing introns

catalytic core is highly conserved across all eukaryotes (we only found spliceosomes in eukarya) and composed entirely of RNA

Question 23

what is SELEX?

Answer 23

systematic evolution of ligands by exponential enrichment (evolution in a test tube)

take pool of random RNA sequences and apply selection pressure via affinity with some other molecule in membrane of column

RT the ones u got and PCR amplify and induce some error w low fidelity DNApol so you get variation and run back through

repeat over and over and eventually you get population of RNAs which bind your ligand of choice w high affinity

Question 24

outline polymerase error rates?

Answer 24

DNA polymerases used in lab (e.g. taq) have varying fidelity; pick whatever one suitable for your SELEX to induce error

can also increase error rates w modified nucleotides; misreading of template = more errors following PCR

Question 25

why do we want to induce error during SELEX?

Answer 25

you want a lot of variants within that population not just original copy

idea is using SELEX to generate RNAs with novel properties

Question 26

how has SELEX been used to develop fluorescent RNAs?

Answer 26

use SELEX approach to select for things that bind fluorogens which are small-molecule dyes that fluoresce by binding aptamers (e.g. RNA)

has allowed discovery of lots of fluorescent RNAs that fluoresce at diff wavelengths and fold into specific structures with lots of non-canonical bping to bind specific fluorogens

can sequence population throughout SELEX to see evolutionary changes e.g. what is conserved as it goes on

Question 27

how was a SELEX-like approach used to develop novel proteins?

Answer 27

one SELEX-like approach called directed evolution used same principles; induce mutations in protein-coding gene, insert into bacteria, select for those with desirable properties, repeat more rounds of this to evolve novel proteins

Question 28

why is RNA structure important?

Answer 28

can be essential for function

Question 29

outline RNA structural components?

Answer 29

RNA structures are module meaning they can be decomposed into subcomponents e.g. loops, stems, bulges - these are what we use for computational modelling

Question 30

how can we represent RNA secondary structure with dot-bracket notation?

Answer 30

if not involved in watson-crick bp you put a dot and if it is you put a bracket

Question 31

what is the difference between canonical (watson-crick) and non-canonical (wobble) base pairs?

Answer 31

canonical - C-G, A-U (3/4 of RNA bp are canonical)

non-canonical - G-U

Question 32

discuss how all base-pairs are possible?

Answer 32

under certain conditions every base pair can form

i.e G will pair with A, C with C etc.

Question 33

how can RNA base-pairing differ based on geometry?

Answer 33

each nucleotide has three possible edges where each nt interaction can occur

• watson-crick edge (most common)
• sugar edge
• hoogsteen edge

these interactions can also occur in cis (normal bp) or trans (both sugars same direction)

this means we have 18 possible pairing relationships based purely on geometry i.e. lots of diverse nt interactions

Question 34

what is the driving force of RNA folding?

Answer 34

base stacking - RNA backbone is negatively charged so bases stack like coins in a roll

non-canonical interactions underrated in their contribution to RNA structure

Question 35

how can we predict RNA structure?

Answer 35

methods range in effort required and accuracy of result; more effort generally means more accuracy

can predict secondary structure from:

1 - computational prediction e.g. free energy minimisation

2 - indirect experimental evidence e.g. chemical/ enzymatic probing

3 - direct evolutionary evidence e.g. comparative analysis

4 - direct structural evidence e.g. x-ray crysto, NMR, C-EM

1 and 2 more accessible, faster and generate more models

3 and 4 more effort but give more information and more accurate models

Question 36

outline how you predict RNA structure from free-energy minimisation (single-seq)?

Answer 36

algorithm that maximises basepairs to find minimal free energy structures; total energy can be computed by summing energies for each structural component (e.g. stacks, loops) - all you have to do is decompose into structural components and enter sequence

calculates most stable secondary structure that corresponds to that sequence; the more negative gibbs free energy the more stable

energies can be looked up in tables derived from melting experiments; ground state (0) assumed to be completely unfolded i.e. no bping

Question 37

discuss the accuracy of minimum free energy (MFE) as a method to predict RNA secondary structure using computational modelling?

Answer 37

accuracy is low and this method sucks because:

energy parameters estimated from non-biological conditions and models extrapolated from limited experiments

fails to account for a variety of things influencing RNA folding e.g. crowding of cellular environment, PTMs, folding kinetics, co-transcriptional folding, transcriptional pausing

also you end up getting a lot of v different structures with similar energy values

but this method is easy

Question 38

why do we use comparative sequence analysis to predict RNA secondary structure from direct evolutionary evidence?

Answer 38

often RNA structure conserved better than RNA sequence

conserved RNA structure indicated from covarying base-pairs (cause negative selection preserves variation that maintains base-pairs) - identify these with deep alignments

alignments can be pasted into RNAalifold which converts covaration measures to pseudoenergies; gives bonuses to stacks supported by covariation and penalises variation that is inconsistent with pairing; combines this with MFEs for each sequence and gives consensus secondary structure prediction

Question 39

compare RNAalifold with AlphaFold2?

Answer 39

alphafold is an AI model that uses similar approach for protein predictions

• build deep sequence alignments
• find covarying sites
• predict global structure

there is currently no alphafold for RNA cause limited solved RNA structures and also RNA folding more complex cause six torsion angles (protein has 2)

Question 40

what is RNA structure probing?

Answer 40

structure-dependent modification of RNAs can impede polymerases

use reagent that covalently modifies either paired or paired bases

map fragments to full-length RNAs to infer features of RNA structure; can tell if the nt is paired or unpaired based on what reagent used

info can then be used to improve or constrain MFE structure predictions

Question 41

outline the benefit of using high-resolution methods for predicting RNA structure?

Answer 41

e.g. X-ray crysto, NMR, cryo-EM

these are ideal as RNA is challenging; flexible ribose+phosphate backbone, weak long-range tertiary interactions, alternative conformations and multiple functional states

Question 41

outline how despite many non-coding variants being associated w disease this area is largely understudied?

Answer 41

up to 90% significant GWAS results lie in non-coding regions; roughly half of these map to introns

large scale screens for disease association often don’t study non-coding SNPs as coding variants are enriched and can test for function much easier

Question 42

outline how redundancy makes it difficult to link ncRNAs to disease?

Answer 42

proteins are generally single copy but many ncRNAs are multicopy with multiple paralogs or pseudogenes

for most nuclear genes both parental copies expressed

because of this ncRNAs robust to variation due to redundancy; hard to knockout w frameshift etc. cause another copy covers for it

exception is 24 mitochondrial RNA genes as maternally inherited so genes single copy

Question 43

why are mitochondrial variants associated with disease?

Answer 43

mitochondrial transfer RNA genes especially susceptible as single copy and maternally inherited i.e. no redundant copies

22 mt-tRNAs and >350 mutations in these reported; phenotype can be complex; same mutation often results in v diff diseases and vice versa

diseases associated with variants in mitochondrial genes tend to affect intensive processes e.g. muscle and brain function

Question 44

why tf would you get mitochondrial donation treatment? What even is it?

Answer 44

if you a carrier of a mitochondrial syndrome and wanna have kids can do this

take donor eggs, take out nucleus leaving maternal mitchondria, put your own nucleus in and use IVF to produce embryo

Question 45

what are snoRNAs?

Answer 45

required for maturing rRNAs; guide covalent modifications of target rRNAs and snRNAs; some may regulate splicing events

two main classes; H/ACA box and C/D box snoRNAs; called this cause carry motifs

motifs: H - ANANNA; C - AUGAUGA; D - CUGA

i.e. H(ANANNA) which is a hinge and an ACA tail

evolutionarily conserved sites imply important interactions

Question 46

discuss our current understanding of snoRNAs?

Answer 46

some have been well characterised in terms of function (people have figured out their targets)

some are orphans i.e. no known targets

about 17% C/D box and 16% H/ACA box snoRNAs are orphans including SNORD116

Question 47

discuss the snoRNA SNORD116?

Answer 47

C/D box orphan that has strong link with prader-willi syndrome which results from a paternal deletion on chromosome 15 i.e. imprinted locus; characterised by weak muscles and developmental issues in newborns; constant hunger in adults and physical deformities

SNORD116 has 29 tandomly repeated copies i.e. multicopy but all located on same part of genome hence why deletion takes them out –> PWS

function and targets of SNORD116 unknown; recent research may have found a SNORD116 target; potentially influences expression of 200 mRNAs

Question 48

what is the difference between major and minor spliceosome?

Answer 48

target different splice sites and comprised of slightly different RNAs (minor has U11 and U12 while major has U1 and U2, both have U5); there is a lot of homology between them tho

diff stages of formation referred to as diff complexes; key one is B complex (tri-snRNP) which is critical for function; forms on the mRNA at site of intron being removed

Question 49

what is MOPDI aka taybi-linder syndrome?

Answer 49

autosomal recessive disorder characterised by developmental issues

caused by mutations in single copy RNU4ATAC gene encoding U4atac/U6atac snRNA leading to decreased formation of tri-snRNP complex resulting in small splicing defect and retention of introns usually removed by minor spliceosome

U4atac and U6atac have long region of complementarity and bind each other to form part of tri-snRNP complex - most variants linked to MOPDI affect formation of stem loop

severe effect; death by age 3

Question 50

why are there so few ncRNAs linked with disease?

Answer 50

non-synonymous changes in proteins are enriched and easier to test

can be difficult to discover mechanisms of ncRNA function

large numbers of paralogs and pseudogenised copies of ncRNAs make identifying variants difficult

exome sequencing (protein-coding exons) has dominated large-scale efforts to connect genetic variation w disease