RNA Maturation, Noncoding RNA, Processing, and Disease

major steps in RNA maturation/processing

transcription (cotranscriptional modification, type of RNA depends on polymerase), end processing (cutting, caps, tails, caps and tails direct where RNA will go), splicing (removing introns), modifications

4 types of introns

group 1 and group 2 - self splicing, everything evolves from these groups
group 3 - tRNA introns and archea, evolved from group 1
group 4 - spliceosomal (mRNA), evolved from group 2
rRNAs will have spacers are not really introns, rRNA will not get put back together after getting cut

RNA exonucleases

enzymes that made cuts from the ends (endonucleases cut internally), 2 main types, specific or quality control, some need specific chemical signal to cut, other only need phosphorylation, some cut both DNA and RNA

RNA exosome

multiprotein complex which degrades RNA for quality control, two nuclease components for degradation and recognition, a third part for opening up the RNA (Mtr4)

rRNA maturation

ribosomal RNA, transcribed as a polycistronic sequence with spacers instead of true introns so that all the pieces can be moved to the biogenesis site together, Pol-I and Pol-III (5S only), have internally and externally transcribed sequences (ITS and ETS)

ribosome maturation

small subunit 18S plus 33 proteins, large subunit 5S (Pol-III), 5.8S, 28S, plus 46 proteins, biogenesis is very energy intensive, so there are also a lot of intrinsic repair mechanisms for it

AAA-ATPases

pulling off proteins off of the ribosomes as the ribosome is getting generated, each protein will have a tail which allows AAA-ATPases to recognize and bind to them

ITS2 processing

Las1/Grc3 comes in and cleaves the pre-rRNA, then the tail is trimmed by Rat1 exonucleases, Mtr4 of the exosome helps align the RNA in the exonuclease

snoRNAs

recruit modifications for rRNAs, contained in Pol-II transcript introns, complex with protein modifiers (snoRNP)

Pol-II transcripts (mRNA)

for all RNA processing, but focus mRNA processing, Pol-II transcripts provide caps to mRNAs, chemical additions to the 5’ end, protect from degradation, help with nuclear exportation, regulate translation

mRNA splicing

done with the mRNA spliceosome, many things that can do wrong during this pathway (alternative splicing), snRNPs are RNA that help orchestrate mRNA splicing, splicing can create circular RNAs that are of interest because they are more stable

cleavage and polyadenylation

processing of the mRNA tail, capping increases tail formation, happen co-transcriptionally, done by a large protein complex, tail is not encoded in template and is added, regular or alternative sequences (usually mostly As)

mRNA modification players

writers - add modifications
readers - detect modifications
erasers - remove modification
disruptions in modifications are linked to metabolic, neurological disorders, cancer

tRNA structure

tRNAs have a very conserved structure, all tRNAs bind CCA adding enzyme, bind EF-Tu to deliver aa-tRNA to ribosome, fit into ribosomal binding site, differ only for adding and recognizing different anticodons (synthetase which recognizes both acceptor stem and anticodon region)

tRNA breaking mechanisms

two main mechanisms, metal dependent or metal independent, cleave the bond between two nucleotides

RNaseP

trim the 5’ leader of the pre-tRNA, endonuclease even though cleaving the end, ribozyme component helps to catalyze, U from RNaseP flips out, probably coordinates to an Mg2+, then cleaves phosphate

tRNA splicing endonuclease (TSEN)

complex which excises introns from tRNA, two structural proteins, two endonucleases, structure originally based on archea, active site has not changed

PCH

known missense mutation in TSEN which causes intellectual development disorder, tested sites of interests to see which one might cause the mutation, mutating and testing for thermostability is a good way to measure/pinpoint impact of mutations

RNA exosome and tRNA

RNA exosome targets unstable/badly made tRNA

tRNA modifications

most modified RNA in cells, on cheat sheet, this field is growing very fast, but hard to study because tRNA are stable and hard to sequence

hot topics in tRNA

fragments (tRFs) - showing emerging roles in disease, epigenetics, inheritance
suppressor tRNAs - mutations causing pre-mature stop codons in mRNA cause around 10% of genetic diseases, use tRNA to fix the stop or substitute the gene

ribozymes

catalytic RNA, catalytic ability is central to idea that RNA is origin of life, mostly rely on Mg2+ but can be metal or nonmetal, usually hydroxyl based mechanisms

hammerhead ribozyme

cleavage on one side of the RNA

HDV

hepatitis delta virus that is a ribozyme, has a pseudoknot structure, can be important structurally for ribozymes but can also cause frameshifting in ribosomes by clogging them

ribozymes as tools

use them to cut RNA in a specific way in lab and in vivo

riboswitches

regulate gene expression, bind a metabolite and change structure to expose an expression domain, not really a catalyst, more like a cofactor, often in 5’-UTR of mRNA

thiamine pyrophosphate riboswitch

widespread in nature, blocks the ribosome binding site, can cause early termination of transcription by stabilizing structure to stop RNA polymerase, since structure is so small, could use NMR to observe structure and conformational switches

long non-coding RNA (lncRNA)

can be transcribed by any polymerase (a little different if Pol-II in the caps ), longer than 200 nt but can range to thousands, help with chromatin modifying complexes, gene expression, development, phase separation

x-inactive-specific transcript (XIST)

lncRNA that plays a role in x-chromosome inactivation, sponge for miRNA, structure gets altered by methylation of an A base, unfolding bases which allows protein to bind

TERC

lncRNA required for telomere maintenance

some miRNA functions and links

linked to cancers, neurodevelopment, viral infection, immune responses, diagnostic markers

drosha

similar to dicer, a ruler for dsRNA hairpins, recognizes GHG (H = anything but G), uses a DGCR8 cofactor

RISC complex

multi protein complex, directs small RNA to gene silencing, usually from DICER or Drosha, argonaute (AGO) plus others, matches guide strand of mi/siRNA to template mRNA, which it cleaves to silence

circRNA

generated mostly from mRNA backslicing, low concentrations in vivo, most studied as miRNA sponges, but definitely biomarkers, of interest for tumor suppressing, hard to detect because they don’t have ends

degradation of circRNA

harder than typical RNA degradation because no ends, but many paths