#3 Ribosome Biogenesis

Ribosome biogenesis

formation of ribosomes, major anabolic activity of cells, highly conserved ribonucleoprotein machinery

Northern blot

pre-RNA processing analysis using affinity tags on protein of interest, purification using binding then cutting of affinity tags leaving intact complexes

Ribosome biogenesis factors (RBFs)

proteins that transiently associate with pre-ribosomes, often essential, may contain RNA binding domains, many different protein families

Energy-requring RBFs

drive ribosome biogenesis directionality , ATP-dependant RNA helicases, ATPases/GTPases, kinases

Nucleoli

membrane-less organelles within nucleus, involved in rRNA synthesis and ribosomal assembly, may each represent individual stages of ribosome biogenesis, requires ongoing rRNA transcription (cancer diagnostic marker), surrounded by shell of heterochromatin (NADs)

Nucleoli FC

Fibrillar centers (FC): rDNA/RNA Pol I

Nucleoli DFC

Dense fibrillar compartments (DFC): nascent rRNAs, ribosomal proteins, assembly factors for early maturation

Nucleoli GC

Granular compartment (GC): pre-ribosomal particles for further maturation

Nucleoli FC/DFC border

likely location of rRNA transcription, contains active rDNA repeats

Phase separation

may occur in nucleoli due to many nucleolar proteins containing intrinsically disordered regions

Nucelear reformation

seeded process dependent on rRNA transcription from NOR, pre-RNA promotes FC -> DFC -> GC formation

Nuclear organizing region (NOR)

rDNA cluster, contains genes for pre-ribosomal compartments

rRNA genes

organized as tandem repeats in 5 clusters of NORs, only ~50% rDNA repeats actively transcribed, different transcriptional states

constitutively silent

heterochromatin (condensed, methylation, histone deacetylation), NoRC important, only upon differentiation (does not occur in ESCs)

Nuclear remodeling complex (NoRC)

binds to pRNA (lncRNAs derived by processing of intergenic sequence transcripts from spacer promoter), establishes heterochromatin confirmation

Transcriptionally permissive rDNA

euchromatin confirmation (decondensed, unmethylated, acetylated), requires further regulation to determine if transcription occurs or not, UBF important

Active Transcriptionally permissive rDNA

UBF (Upstream binding factor) binding, Pre-initiation complex formation, no nucleosomes in coding region

Inactive/poised Transcriptionally permissive rDNA

nucleosomes in coding regions, repressive histone modifications

RNA Pol I transcription

major regulatory control point for ribosome biogenesis, rate limiting factor, long transcript

Epigenetic regulation of RNA Pol I txn

occurs in response to cellular conditions, constitutively silenced vs. transcriptionally permissive rDNA

Regulation at initiation of RNA Pol I txn

pre-initiation complex must be assembled, TF modulation

-PIC: pre-initiation complex, requires UBF binding to promoter, recruitment of SL-1 complex and RRN3-mediated binding of RNA Pol I

-TF modulation: growth factors/nutrient availability/energy affect TFs, regulating initiation of RNA Pol I transcription "

Pre-rRNA processing

pre-rRNA precursor processed into mature 18S, 5.8S, 28S rRNA, pathway well-conserved, occurs in cytoplasm

separation cleavage - human cells, pre-40S + pre-60S formation

Pre-rRNA modification

extensively modified via base methylations (methyltransferases) or snoRNPs (ribose methylation, pseudoruidylation), affect ribosome structure/function/interactions, redundancies can prevent defects

SnoRNPs

"small nucleolar RNPs, contain snoRNAs that act as guides via base-pairing for modifications

-Box C/D catalyze ribose methylations, Box H/ACA catalyze pseudouridylations"

Co-transcriptional compaction

pre-rRNA is folded as it is transcritped from DNA via associations of RPs/ribosome biogenesis factors

snoRNP modifcations

occur within nucleosome as rRNA begins to be transcribed, binding of proteins helps prevent misfolding

SSU proceosome assembly

small subunit proceosome, r-proteins associate to support 18S rRNA folding

LSU proceosome assembly

large subunit proceosome, r-proteins associate starting in nucleosome, continues outside of nucleosome

60S folding

occurs in domains (I/II/VI -> III/V -> V/IV)

5S rRNP

association of 5S rRNA and uL18/uL5, mediated by Rpf2-Rrs1 (but must leave for final conformation)

Nuclear export of pre-ribosomes

pre-ribosomes exported as individual subunits, multiple nuclear export receptors/export factors recruited after final maturation

Yeast Nuclear export pre-ribosomes

-Nmd3: nuclear export sequence containing adaptor for Crm1

-Mex67/Mtr2: nuclear export factor but for pre-60S export, interact with nucleoporins "

Human nuclear export pre-ribosomes

-NMD3: NES-containing adaptor for Crm1

-EXP5: RanGTP-dependant export receptor, supports 60S export

-40S export: requires multiple copies of CRM1 with multiple adaptor proteins

Cytoplasmic maturation

final pre-rRNA processing, assembling of late RPs, release of biogenesis  factors, proofreading of functional centers, requires energy-driven step-wise activation/recruitment of proteins

Ribosomal proteins (RPs)

rapidly synthesized/transported/assembled into pre-ribsomes , interact with rRNA, affects folding, creates bidning sites for other proteins

Ribossomopathies

diseases linked to genes in ribosome protein or ribosome biogenesis, often affects blood, have higher chance of cancer