RNA PROCESSING

RNA capping the first modification of premRNA

RNA pol II produce 25 nts of RNA the 5 end of the new RNA molecule is modified by addition of the cap 7 methyl guanosine. Three enzymes acting are
1. 2. 3.

Phosphatase- removes phosphate from 5 end of nascent RNA. Guanyl transferase- adds GMP in the reverse linkage (5 to 5) Methyl transferase- adds methyl group to guanosine

5 cap

It distinguishes other RNA present in the cell. Cap binds a protein complex called CBC (cap-binding complex). It helps the RNA to properly processed and exported. It has important role in translation of mRNAs in the cytosol

RNA pol II pauses and the kinase positive transcription elongation factor b (P-TEFb) phosphorylates RNA pol II on the serine 2 residue in the repeat unit to C-terminal domain (CTD) of the large subunit of the enzyme. Synthesis of the cap is carried out by the enzymes tethered to the CTD of pol II. The cap remains tethered to the CTD through an association with the capbinding complex (CBC)

P-TEFb is composed of cyclin dependent kinase (CDK9) and either cyclin T1, T2 or K. This terminal is also called Cterminal domain kinase1 (CTDK1). The pausing and regulatory phosphorylation event allows for the potential of attenuation in the rate of transcription.

5 Cap
Type 1 cap: the ribose (O2) gets methylated (and the first base if A-N6 gets methylated). Type II cap: in some species the subsequent residue at +2 position is also methylated again at O2 of ribose

The capping reaction can be used to regulate the protein synthesis, a strategy utilized by some animals during egg maturation. Most RNA viruses cap their genomes and mRNAs whilst the Picornaviridae, whose infection strategy exploits their lack of cap dependence, block the 5 end of their genome with a viral protein. The orthomyxoviridae do not cap their genome segments but steal pre-formed caps from the host mRNAs, a transesterification process which has been termed capsnatching

Polyadenylation of mRNA

A specific sequence is recognized by the CPSF endonuclease activity of the polyadenylate polymerase (PAP) which cleaves the primary transcript at 3 end of the mRNA. Initial polyadenylation is slow because PAP dissociates after adding each adenylate residue. After synthesis of short stretch the PABP attaches to the tail and increases the processivity A stretch of 20-250 A residues is then added to the 3end by the polyadenylate polymerase activity.

The mRNA site where cleavage occurs is marked by the two sequence elements The conserved sequence 5AAUAAA3 upstream 10-30 nts on 5 site(cleavage site). Sequence rich in G and U residues, 20-40 nts downstream of the cleavage site.

i. CPSF

(cleavage and polyadenylation specificity factor). ii.CstF (cleavage stimulation factor). iii.Two cleavage factor proteins (CFI and CFII).
After

cleavage, the enzyme poly(A) polymerase (PAP) adds A nucleotides to the 3 end of the RNA, using ATP as a substrate. PAP is bound to CPSF during this process. PABII (poly(A) binding protein II) binds the poly(A) tail as it is produced

Histone mRNAs and the genomes of certain plant viruses are not polyadenylated. A secondary structure adopted by the histone transcripts is responsible for the 3 end maturation, which involves U7 snRNA and associated proteins The poly (A) tail and its associated proteins probably help protect mRNA from enzymatic destruction. Many bacterial mRNA also acquire poly(A) tails, but these tails stimulate decay of mRNA rather than protecting it from degradation.

INTRONS

Introns are removed by a process called splicing. Present in higher organisms more often than lower organism. All the 3 classes of RNA has introns in eukaryotes. Very few introns are known in bacteria

INTRON

Initially described in Adenovirus and later in the ovalbumin gene of the chicken

The isolated ovalbumin gene was denatured and rehybridized with mRNA from a chicken egg The hybrids were examined using electron microscopy D loops formed, representing single stranded regions of genomic DNA not present

Self splicing introns

First releaved in 1982 in the studies of splicing mechanism of the group I rRNA intron from the ciliated protozoan Tetrahymena thermophila, conducted by Thomas Chech and colleagues. They transcribed isolated tetrahymena DNA including intron in vitro using purified bacterial RNA polymerase. The resulting RNA spliced itself accurately without any protein

Self- splicing intron

The 2 most common self-splicing mechanism are

1.

2.

group I intron group II intron

Group I intron are found in nuclear, mitochondrial and chloroplast genes. Group II in mitochondrial and chloroplast mRNA in fungi, algae and plants primary transcript. Many of the group I and group II are self splicing and do not require ATP hydrolysis.

Group I

It requires external guanosine nucleotide as a cofactor. The 3-OH of the guanosine nucleotide acts as a nucleophile to attack the 5 phosphate of the 5 nucleotide of the intron and covalently attaching the two exons together. The spliced intron is eventually degraded.

Group II

Group II intron are spliced (without external nucleophile) the 2 OH of Adenine residue within the intron. This residue attack the 3 nucleotide of the 5 exon forming an internal loop called a lariat structure. The 3 end of the 5 exon then attacks the 5 end of the 3 exon as in group I splicing releasing the intron and covalently attaching the two exons together.

Splicing by spliceosome (Group III)

Here the splicing is catalysed by specialised RNAprotein complexes called small nuclear ribonucleoprotein particles (snRNPs) The RNAs found in snRNPs are identified as U1(165bs), U2(188bs), U4(142bs), U5(116bs) and U6(107bs). The genes encoding these snRNAs are highly conserved in vertebrate and insects and are also found in yeasts and slime moulds indicating their importance. Spliceosome introns generally have the dinucleotide sequence GU at 5 end and AG at 3end.

The U1 RNA has sequences that are complementary to sequences near the 5 end of the intron. The binding of U1 RNA distinguishes the GU at 5 end of the intron from other randomly placed GU sequences in mRNAs. The U2 RNA also recognizes sequences in the intron, in this case near the 3 end (branch point). The addition of U4, U5 and U6 RNAs forms a complex spliceosome. This then removes the intron and joins the two exons together.

The U1 snRNP forms base pairs with the 5 splice junction, and the BBP and U2AF recognise the branch point

The U2 snRNP displaces BBP and U2AF and forms base pairs with the branch point site consensus sequence.

The U4/U6-U5 triple snRNP enters the reaction. In this triple snRNP the U4 and U6 snRNAs are held firmly together by base pair interactions

Subsequent rearrangements create the active site of the spliceosome and position the appropriate portions of the pre-mRNA substrate for the first phosphoryl-transferase reaction.

Several more RNA-RNA rearrangements occur that break apart the U4/U6 base pairs and allow the U6 snRNP to displace U1 at the 5 splice junction to form the active site for the second phosphoryl transferase reaction, which completes the splice.

SPLICEOSOME USES ATP HYDROLYSIS

The exchange of U1 snRNP for U6snRNP occurs before the first phosphoryl-transfer reaction. This exchange requires the 5 splice site to be read by two different snRNPs thereby increasing the accuracy of 5 splice site selection by the spliceosome.

SPLICEOSOME USES ATP HYDROLYSIS

The branch point is first recognised by BBP and subsequently by snRNP, this check and recheck strategy provides increased accuracy of site selection. The binding of U2 to the branch point faces the appropriate adenine to be unpaired and thereby activates it for the attack on the 5splice site.

SPLICEOSOME USES ATP HYDROLYSIS

After the first phosphoryl-transfer reaction has occured a series of rearrangement brings the two exons into close proximity for the second phosphoryl transfer reaction. The snRNAs both position the reactants and provide the catalytic sites for the two reactions. The U5 snRNP is present in the spliceosome before this rearrangement occurs.

Regulatory elements in pre mRNA

Cis-regulatory elements in pre-mRNA splicing. Information in pre-mRNA substrate contributing to the splice site recognition includes short and consensus sequences at 5 splice site(ss), 3 branch point site(BPS) which are typically located 30-50 nts upstream of the 3 ss in human. Polypyrimidine tract (PPT) is just downstream of the BPS. But this is short sequence and relatively degenerative. Additional flanking cis elements in pre-mRNA are required to facilitate splice site recognition and selection. Based on the position and function these cis elements are divided into 4 categories. ESEs, ESSs, ISEs, ISSs. They serve as binding sites for trans regulatory factors, such as members of SR and hnRNP protein families, which in turn regulate splicing by either promoting or preventing the recruitment of basal splicing machinery.

Alternative splicing are regulated

Splicing factors bind to exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS) to regulate splicing.
1. 2.

Splicing enhancers are recognized by SR proteins. Splicing silencers are recognized by hnRNPs: (it lack SR domain)

The ultimate alternative splicing decisions are therefore made by combinational effects of the similar (synergic) or opposing (antagonistic) splicing regulatory signals.

For short introns (<200-250 nt), spliceosome initially assembles across introns in sequential manner as described above. In case of the long intron (>250), spliceosomal components first assemble across and exon, a process called exon definition. Exon definition:- U1 snRNP binds to the 5 ss downstream of the exon, components of U2 snRNP associate with the polypyrimidine tract/3 ss and BPS upstream of the exon, respectively. Regulatory sequences within the exon recruit protein factors such as the SR protein family members, which bridge and cross exon interaction and stabilize the exon definition complex. Since catalytic steps of the splicing take place across an intron, the cross exon complex must be switched to cross intron complex, a process that is currently not well

Pre mRNA splicing by major spliceosome

ESE - exon splicing enhancer sequences SR ESE binding proteins

Accuracy of splice-site selection

1. Co-transcriptional

loading: factors bound to the 5 site are poised to interact with the factors binding to the next 3 site 2. SR (serine arginine rich) proteins bind to Exonic Splicing Enhancers and recruit the splicing machinery. They ensure that splice sites close to exons are recognized preferentially. SR proteins not only ensure the accuracy and efficiency of constitutive splicing, but also regulate alternative splicing.

Coordination of splicing and transcription provides an attractive mechanism for bringing the two splice sites together

SPLICING ERRORS

Exon skipping: very fast transcription rate and/or inefficient tethering could cause exon skipping. Cryptic splice site: cryptic splicing signals are nucleotide sequences of RNA that closely resemble true splicing signals

Alternate splicing

Alternative slicing is an important layer of gene expression control and enhances the proteomic diversity. Recent studies show that more than 94% humans genes undergo AS events (Pan et al.2008; Wang et al.2008) mRNA transcripts produce only one mature mRNA and one corresponding polypeptide. Others can processed in more than one way to produce different mRNAs and thus different polypeptide. AS regulations have been demonstrated to play pivotal roles in different cell types, developmental stages, across tissues, sex determination and in response to external stimuli.

I. Alternative splicing
Poly (A) site choice

If there are more than one cleavage site and polyadenylation, This use of one closest to the 5 end will remove more of the primary transcript sequence.

II. Alternative splicing

Alternative splicing patterns produce form a common primary transcript 3 different forms of the myosin heavy chain at different stages of fruit fly development.

Both mechanism I and II

This results in diversity in the variable domains of immunoglobulin heavy chains. The recombination of the V & J gene segments of the human IgG kappa light chain.

The primary transcript has the molecular signal for alternate splicing and this pathway is determined by the processing factors, RNA binding proteins that promote one particular path. The basic AS patterns can be classified into
1. 2. 3. 4. 5. 6. 7.

Cassette exon inclusion or skipping. Alternative 5 splice site Alternative 3 splice site Intron retention Mutually excusion alternative exons Alternative promoter and first exon Alternative poly A site and last exon

Additional patterns of alternative splicing

Genes can adopt single or combinational patterns to generate functionally distinct isoforms. It has been estimated that 80% of the AS events affect ORFs, leading to aminoacid deletion/insertion or frame shift. The remaining 20% falls within untranslated regions and influence cis regulatory elements that control mRNA stability, localization and translational efficiency (including miRNA binding sites). Besides a large proportion (1/3rd) of AS events introduced premature stop codons (PTC) which could potentially trigger mRNA degradation via nonsense mediated decay (NMD) pathway

AS is generally controlled by cooperative interplays between RNA binding proteins (RBPs), regulatory elements in nascent transcripts and the basal splicing apparatus. Splicing regulating RBPs bind directly to splice sites, or interact with specific sequences in pre-mRNA to facilitate or block the recruitment of splicing machinery, which in turn stimulate or repress splice site usage(modulating alternative splicing)

AS choices occur at different stages of the splicing process.

Splice site recognition:understood by splicing regulation of survival of motor neuron (SMN) exon7. Splice site pairing:prevention of Fas (CD95) exon 6 inclusion by RNA binding motif protein 5 (RBM5) Combinational effect of both : polypyrimidine tract binding protein

Temporal control of alternate splicing

Influenza A M1 mRNA

Early: Splicosome recognizes M3 splice sitemakes M3 mRNA Late: Viral P proteins recruit cellular SR protein, directing splisosome to M3 splice site to make M2 mRNA.

Tissue specific splicing regulators

1. 2. 3. 4. 5. 6.

Presence of NOVA1 and NOVA2 (Ule et al.2006,2005) nPTB (Boutz et al. 2007) FOX1 and FOX2 (Gehman et al. 2011; yeo et al. 2009) RBM20 (Guo et al.2012) RBM35(Warzecha et al. 2009) RBM11 (pedrotti et al. 2011)

Ubiquitously expressed splicing regulators also participate in the tissue specific alternative splicing regulation. Differentially expression of tissue specific splicing regulators and /or variable concentrations and/or modifications of ubiquitously expressed splicing regulators will also regulate the tissue specific splicing regulators.

Altered chromatin structure and AS control

Altered chromatin structure affected the transcription rate, thereby changed the decision of AS. It has recently been shown that histone H3 trimethylated at lysine residue 36 (H3K36me3), a marker for active transcription, specifically marks exons and occurs more frequently on constitutive exons compared with alternative exons. These suggest a kinetic mechanism for transcription coupled alternative splicing control.

Exon shuffling

Alternative splicing generates multiple products from a single gene new genes can be created by exon shuffling. Many genes (and their products) have arisen via exon duplication and divergence. Intron/exon size ratio ensures recombination within the introns. Alternative splicing allows new exons.

Alternative splicing and human disease

Defects in splicing are regarded as a primary cause of diseases and mechanisms of which can be classified into two main groups
1.

2.

Disruption of cis elements: splice sites, splicing cis regulatory elements. Disruption of trans acting factors : component of core splicing machinery and splicing regulators.

Cis effects: disruption of splicing code

They are inherited human disorders. These mutations cause

1.

2.
3.

Change in encoded protein Alter the ratio of natural protein isoforms Premature termination codon

Duchenne muscular dystrophy (DMD) mutation in massive dystrophin gene. Ex:- TA substitution in exon 31 simultaneously create a premature stop codon and an exonic splicing silencer(ESS), leading to exon 31 skipping and mild form of DMD. Frontotemporal dementia and parkinsonism linked chromosome 17 (FTDP17), which is an autosomal dominant disorder caused by mutations in the MAPT gene that encodes tau.

Numerous point mutations within and around MAPT exon 10 destruct the exonic or intronic splicing elements and alter normal 1:1 ratio of isoforms with or without exon 10. The disrupted splicing in turn destroys the balance between tau proteins containing either four or three microtubule-binding domains (R) and causes FTDP17.

Trans effects: defects in splicing machinery/regulators

Mutation or stoichiometric changes in these regulators can extensively change the AS patterns of their targets. Autosomal dominant form of retinitis pigmentosa (adRP) that caused by mutations in five proteins belonging to U4/U6-U5 tri-snRNP:PPRF31, PPRF8, PPRF3, RP9 and SNRNP200. Another example is spinal muscle atrophy (SMA) that cause the mutations in SMN gene which plays a complex role in snRNA biogenesis. It is found that snRNA is affected differently in distinct tissue of SMN-deficient mouse rather than a uniform decrease. Point mutation in U4atac snRNA (component of minor spliceosome) linked with microcephalic osteodysplastic primordial dwarfism type I (MOPD-I i.e.,Taybi-Linder syndrome)

mRNA stability

The concentration of any molecule depend on Rate of synthesis and rate of degradation. Change in steady state may accumulate or deplete the mRNA. Average half life of a vertebrate mRNA 3hrs, with the pool of each type of mRNA turning over about 10 times per cell generation. Half life of bacterial mRNA is about 1.5min.

mRNA s are degraded by ribonucleases. In E.coli many cuts mRNA by endonuclease followed by 3 to 5 degradation by exonuclease. A hairpin structure in bacterial mRNAs with a rho-independent (hairpin) confers stability against degradation.

In lower eukaryotes the major pathway involves first shortening the poly(A) tail, then decapping and degrading the mRNA in the 5-3 direction. Higher eukaryotes

3-5 degradation is major pathway in higher eukaryotes. All eukaryotes have conserved 10 exosome (3 5 exonuclease) involved in processing of 3 end of mRNAs, rRNAs, tRNAs, snRNAs and snoRNAs.

Control of mRNA stability

mRNA for milk casein has half life of 1 hrs. When stimulated with prolactin half life increases to 40 hrs.

tRNA processing

In E.Coli some tRNA genes are co-transcribed with rRNA genes in a common operon.

Ribonuclease P cleaves at the mature 5 end of such transcripts. Ribonuclease D processes the 3 end exonucleolytic degradation. tRNA nucleotidyl transferase CCA is added to the 3 end of the tRNA.

In Eukaryotes and Archaea, some tRNA has introns which are further removed.

tRNA splicing

These introns are spliced by a specific splicing endonuclease that involves a cut and paste mechanism. In order for tRNA intron removal to occur the tRNA must first be properly folded into its characteristic overleaf shape. Misfolded precursor tRNAs are not processed which allows the splicing reaction to serve as a control step in the generation of mature tRNAs.

tRNA processing

Endonuclease P tRNA nucleotidyltransferase.

Modified bases in tRNA

10% of nucleotides become modified during tRNA synthesis usually by posttranscriptional chemical modification.

Eukaryotic rRNA Processing

The 45S precursor is methylated at more than 100 of its 14,000 nucleotides, (bases or 2-OH) (Uridines pseudouridine) Enzymatic cleavage of the 45S precursor produces the 18S, 5.8S and 28S rRNAs and assembles with ribosomal proteins. All processing require small nucleolar RNAs (snoRNA) found in protein complexes (snoRNPs) in the nucleolus that are reminiscent of spliceosomes. The 5S rRNA is produced separately

Bacterial rRNA processing

Special function RNAs undergo processing

snRNAs & snoRNAs are synthesised as large precursors and then processed. Some snoRNAs are encoded within the introns of other genes. After splicing snoRNP binds to snoRNA and remove extra RNA at both ends. Pre-snRNA are synthesised by RNA pol II, ribonuclease remove extra RNA at each end. snRNA undergo modification.

The end