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Section O – RNA processing and RNPs. Contents. O1 rRNA processing and ribosomes Types of RNA processing , rRNA processing in prokaryote , rRNA processing in eukaryotes , RNPs and their study , Prokaryotic ribosomes , Eukaryotic ribosomes O2 tRNA processing, RNase P and ribozymes
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Contents O1 rRNA processing and ribosomes Types of RNA processing, rRNA processing in prokaryote, rRNA processing in eukaryotes, RNPs and their study, Prokaryotic ribosomes, Eukaryotic ribosomes O2 tRNA processing, RNase P and ribozymes tRNA processing in prokaryotes, tRNA processing in eukaryotes, RNase P, Ribozymes O3 mRNA processing, hnRNPs and snRNPs Processing of mRNA, hnRNP, snRNP particles, 5’ Capping, 3’ Cleavage and polyadenylation, Splicing, Pre-mRNA methylation O4 Alternative mRNA processing Alternative processing, Alternative poly(A) site, Alternative splicing, RNA editing
O1 rRNA processing and ribosomes — Types of RNA processing • Very few RNA molecules are transcribed directly into the final mature RNA. Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product. RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.
Cytoplasm Nucleus or Nucleolus primary transcript RNA processing Romoval of nucleotides addition of nucleotides to the 5’- or 3’- ends modification of certain nucleotides mature RNA.
(1) Removal of nucleotides by both endonucleases and exonucleases • (2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products. • (3) Modification of certain nucleotides on either the base or the sugar moiety.
pre-5S rRNA Pre-16S rRNA Pre-tRNA Pre-23S rRNA Pre-tRNA Promoters Terminators O1 rRNA processing and ribosomes — rRNA processing in prokaryote • There are 7 different operons for rRNA that are dispersed throughout the genome. • Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons. • The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived. rRNA operon
Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences RNA folding
Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome RNP complex formation
Step 3: After the binding of proteins, nucleotide modifications take place. Example: methylation of adenine by methylating agent S-Adenosylmethonine (SAM) Step 4: RNA cleavage
pre-5S rRNA Pre-16SrRNA Pre-tRNA Pre-23S rRNA Pre-tRNA Promoters Terminators Transcription 30S pre-rRNA: RNase III III P F III III P F P E Cleavage at RNase M16 M16 M23 M23 M5 16S rRNA tRNA 23S rRNA 5S rRNA tRNA rRNA operon
O1 rRNA processing and ribosomes — rRNA processing in eukaryotes • rRNA in eukaryotes is also generated from a single, long precursor molecule by specific modification and cleavage steps • The processes are not so well understood
The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol I Precursor sizes are different among organisms (yeast: 7000 nt; mammalian 13500 nt), and pre-mRNA processing is also slightly different among organism.
3. The precursor contains • one copy of the 18S coding region and • one copy each of the 5.8S and 28S coding regions, which together are the equivalent of the 23S rRNA in prokaryote 4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.
5. The eukaryotic 5S rRNA • is transcribed by RNA Pol III from unlinked genes to give a 121nt transcript • the transcript undergoes little or no processing
18S 5.8S 28S 47S ETS1 ITS1 ITS2 ETS2 45S 41S 20S and 32S Mature rRNAs 18S rRNA 5.8S rRNA 28S rRNA Mammalian pre-rRNA processing Indicates RNase cleavage
The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced. • Mature rRNAs complex with protein to form RNPs (nucleolus) • Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)
Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced out to generate mature rRNAs. • Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme
O1 rRNA processing and ribosomes — RNPs and their study • Cells contain a variety of RNA-protein complexes( RNPs). • These can be studied using techniques that help to clarify their structure and function. • These include dissociation, re-assembly, electron microscopy, use of antibodies, RNase protection, RNA binding, cross-linking and neutron and X-ray diffraction. • The structure and function of some RNPs are quite well characterized.
O1 rRNA processing and ribosomes — Prokaryotic ribosomes • Protein biosynthetic machinery • Made of 2 subunits (bacterial 30S and 50S, & Eukaryotes 40S and 60S) • Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes • In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.
Features of the E.coli ribosome Cleft Platform Central protuberance Stalk Small
Ribosome Structure (2) • mRNA is associated with the 30S subunit • Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits. • The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.
O1 rRNA processing and ribosomes — Eukaryotic ribosomes • larger and more complex than prokaryotic ribosomes, but with similar structural and functional properties
O2 tRNA processing, RNase P and ribozymes — tRNA processing in prokaryotes • Mature tRNAs are generated by processing longer pre-tRNA transcripts, which involves • specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by • base modifications which are unique to each particular tRNA type.
O2 tRNA processing, RNase P and ribozymes — tRNA processing in eukaryotes • The pre-tRNA is synthesized with a 16 nt 5’-leader a 14 nt intron and two extra 3’-nucleotides.
Primary transcripts forms secondary structures recognized by endonucleases • 5’ leader and 3’ extra nucleotide removal • tRNA nucleptidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end • Intron removal
O2 tRNA processing, RNase P and ribozymes — RNase P • Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors • RNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs) • In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa. • RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.
O2 tRNA processing, RNase P and ribozymes — Ribozymes • Ribozymes are catalytic RNA molecules that can catalyze particular biochemical reactions. • RNase P RNA is a ribozyme. • RNase P RNA from bacteria is more catalytically active in vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures. • Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences • Group I introns, such as Tetrahymena intron • Group II introns.
Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA • HDV ribozyme • Hairpin ribozyme • Hammer head ribozyme • Ribozymes can be used as therapeutic agents in • correcting mutant mRNA in human cells • inhibiting unwanted gene expression • Kill cancer cells • Prevent virus replication
O3 mRNA processing, hnRNPs and snRNPs — Processing of mRNA • Processing of mRNA: prokaryotes • There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed. • Prokaryotic mRNA is degraded rapidly from the 5’ end • Processing of mRNA in eukaryotes • In eukaryotes, mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA). • Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs
O3 mRNA processing, hnRNPs and snRNPs — hnRNP • The hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) • The hnRNP proteins are though to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions
O3 mRNA processing, hnRNPs and snRNPs — snRNP particles • snRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs. • The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6. • A large number of snRNP define methylation sites in pre-rRNA. • snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap. • Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins. • Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.
O3 mRNA processing, hnRNPs and snRNPs — 5’ Capping • Very soon after RNA Pol II starts making a transcript, and before the RNA chain is more then 20 -30 nt long, the 5’-end is chemically modified. • 7-methylguanosine is covalently to the 5´ end of pre-mRNA. • Linked 5´ 5´ • Occurs shortly after initiation
Function of 5´cap • Protection from degradation • Increased translational efficiency • Transport to cytoplasm • Splicing of first exon
O3 mRNA processing, hnRNPs and snRNPs — 3’ Cleavage and polyadenylation • In most pre-mRNAs, the mature 3’-end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail. • RNA polymerase II does not usually terminate at distinct site • Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA) • ~250 AMPs are then added to the 3´ end • Almost all mRNAs have poly(A) tail
Function of poly(A) tail • Increased mRNA stability • Increased translational efficiency • Splicing of last intron
O3 mRNA processing, hnRNPs and snRNPs — Splicing • the process of cutting the pre-mRNA to remove the introns and joining together of the exons is called splicing. • it takes place in the nucleus before the mature mRNA can be exported to the cytoplasm. • Introns: non-coding sequences • Exons: coding sequences • RNA splicing: removal of introns and joining of exons • Splicing mechanism must be precise to maintain open reading frame • Catalyzed by spliceosome (RNA + protein)
Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule.The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.
Lariat C U R A Y
Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.
Spliceosome • Catalyzes pre-mRNA splicing in nucleus • Composed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the pre-mRNA being assembled • U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity. • U2 and U6 snRNA are able to catalyze the splicing reaction.