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LET'S TALK LIFE SCIENCE MOLECULAR BIOLOGY

Suraj Prakash Sharma | Ekta Chotia

SPLICING
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4.            SPLICING

Splicing is a process of removing introns and ligating exons. RNA has two type of sequences one is translated into protein called as exon and other is not encoded for any protein called as introns. The transcript formed after transcription of DNA is called as primary transcript. The primary tanscript undergo splicing. Primary transcript is also known as hnRNA (heterogeneous nuclear RNA). It is a precursor of mRNA.

Splicing occur in both prokaryotic and eukaryotic. Introns are more variable than exon. Intron size is larger than exon size. Mutation in introns does not affect the protein structure because introns are spliced out during splicing. If mutation occurs in exon then it affect the protein structure, incase that protein crucial for cell survival in turn cell death occur, thus selection pressure always on exon.

Splicing process occur in nucleus of eukaryotic cell. .

Intron is interspersed between exons throughout the precursor of m-RNA, rRNA and tRNA. tRNA introns are relatively small (average size is 4 - 50 base). eukaryotes two type of intron are present (i) the intron boundary is made by GU-AG and (ii)  the intron boundary is made by AU-AC. These intron boundary has 100% conserved sequenced.

In GU-AG intron GU at 5¢ end and AG at 3¢ end are present. Similarly in AU-AC intron AU at 5¢ end AC at 3¢ end are 100% conserved sequence. GU-AG and AU-AC intron also known as spliceosomal introns.

4.1.         Structure of GU-AG intron

In GU-AG intron, the GU is present at 5' end of splice site and AG is present at 3' end of intron. A polypyrimidine tract (C and U) is present at upstream of 3' splice site. And upstream of polypyrimidine tract  a conserve sequence CURAC is present. The R denotes the purine nucleotide. The adenine in CURAC sequence is highly conserved. The adenine is called as branch point.

If point mutation occure in DNA or some error occur during transcription, a "cryptic splice site" is formed in mRNA and this part is not spliced out. The mature mRNA formed after splicing in this case is different from normal mRNA. Thus a single point mutation leads to deletion in final protein product.

Mechanism of GU-AG intron splicing :

First Step is the formation of lariat shaped intermediate. This lariat forms when 2'-OH of Adenine of branch point attacks the phosphodiester bond between the first nucleotide of intron G and last nucleotide of exon. This is known as first transesterification reaction. Thus first nucleotide of intron (G) form 5' - 2' phosphodiester bond with branch point Adenine. Second transesterification reaction carried out by free 3’OH of exon at 5’ splice site. The 3'OH attack on phosphodiester bond between the 1st nucleotide of exon at 3' splice site nucleotide of Guanine of intron at 5' splice site. Thus this lariat intron structure remove and 3’OH of exon first ligated with 5’ phosphate of exon two ATP also involve for assembly of splicing complex.

Type of Splicing

4.2.         Major Splicing :

Splicing of intron carried out with the help of spliceosome, a large molecular apparatus. Spliceosome contain 5 snRNA U1, U2, U4, U5 and U6 and 150 protein, each of them having some specific role.

SnRNA are small nuclear RNA, confined to nucleus of cell. They are involved in RNA processing. When SnRNA associates with proteins they are called as SnRNA (SnurP).

Each SnKNP contains a single SnRNA and several (< 20) proteins. The U4 and U6 SnRNPS are made up of one RNA and one protein only.

All SnRNA has a conserve sequence PUAU36GpU. The Sm proteins bind to this conserved sequence.

This sequence in absent in U6. The U6 SnRNA has LSm proteins. LSm proteins are Sm Like protein.

Cajal body site of post transcription modification of snRNA and snRNPs assembly. This splicing also called canonical splicing.

Other protein like RNA-annealing factor, Which help in loading of snRNPs onto mRNA, BBP/SF2 (branch binding protein / supporting factor), DEAD-box helicase protein, use ATPase activity to remove RNA:RNA interaction, allow different RNA:RNA pair to form, thus rearrangement with in splicing complex occur.

The U1 RNPs recognize the 5' splice site and bind to it by RNA:RNA interaction. i.e. The U1RNA recognise the 5' splice site. Another protein factor is called as U2AF (U2 auxilary factor)-SR protein family member rich in serine-argenine region. It contain two domain 65 and 35 which binds to intron. The U2AF-65 domain bind at pyrimidins track and U2AF-35 domain bind at AG the 3’ splice site intron boundary by protein-RNA interaction. BBP/SF2 (branch binding protein / supporting factor) forming bridge between U1SnRNA and U2AF by binds to both of them. This complex now called E complex (early complex) also known as commitment complex.

Then U2SnRNA binds at branch point. This complex knows as A complex or prespliceosome complex. U2 SnRNA bind at branch point with ATP in such a way that result in extrudance of Adenine from is position now this unpaired Adenine available to act on 5’ splice site.

Now U5SnRNA, U4SnRNA and U6SnRNA come altogether called tri-snRNP particle. The U5SnRNA bind in loose association with exon at 5’ splice site and U6SnRNA and U4SnRNA bind on intron at upstream of CURAC sequence and complex become B1 complex. This complex is also called as spliceosome.

U6 snRNp and U4 snRNp attached to each other by  extensive base-pair interaction by stem loop structure. Prp24 and LSms protein associated with free U6 snRNp the Prp24 form an intermediate complex with the U6snRNA, and responsible for extensive base-pairing between the U4 SnRNP and U6 snRNAs. The  LSms protein support U6 snRNP in this function.

Then ATP bind to U2 SnRNA get hydrolyzed, result in U1 SnRNP dissociation and movement of U5 SnRNP at intron and binding of U6 SnRNP at GU. This complex called as B2 complex. U4 SnRNP now dissociate and thus U5 SnRNA is move at 3' splice site.

U6 SnRNA has endonuclease activity. This break the phosphodiester bond at 5' splice site. Now Guanine of 5' splice site bind with Adenine branch point by the forming of 5’-2’phosohodiester bond and thus lariat structure formation occur. And this complex called C1 complex. This first transestrification reaction is done by U2 SnRNP and U6 SnRNP, attached to each other by base paring and form active site within the mention reaction occur.

U5 SnRNP break phosphodiester bond between Guanine and Cytocine and makes phosphodiester bond between two nucleotide of Exon 1 and Exon 2. A second transesterification reaction done by U5 (has endonuclease activity). Thus lariate dissociate and two exons ligated together.

4.3.         Minor Splicing

Splicing of AU-AC intron is known as minor splicing. This is also called an U12 type splicing. The splicing of AU-AC intron has similar process as occur in major splicing. In minor splicing. The role of U1 SnRNP, U2 SnRNP, U4 SnRNP, U6 SnRNP is performed by U11 SnRNP, U12 SnRNP, U4atac SnRNP and U6atac SnRNP respectively and U5, BBP/SF1, CURAC, polypyrimidine track represent as same as major splicing. The minor splicing also occurred in nucleus and also known as noncanonical splicing. e.g., this splicing occurs in insects, eukaryotic mRNA in plant, vertebrates , fungi (Rhizopus oryzae) and in some human gene performing essential cellular functions.

4.4.         Auto splicing

RNA could splice themselves without any protein called ribozyme. Thomas Cech found auto-splicing in 26S  pre- rRNA gene of Tetrahymena , a ciliated protozoan. Auto splicing occurs by RNA of intron that use same spliceosome mechanism to remove intron without the help of protein only through RNA themselves with the involvement of ATP.

There are three type of autosplicing.

(i)            Group-I

(ii)           Group-II

(iii)          Group-III

4.4.1.     Group-I

Group I autosplicing is found in nuclear, mitocondrial and chloroplast genes of m-RNAs, r-RNAs, t-RNAs. It has nine paired region (P1-P9) with in its core secondary structure. This paired region folded into two domain one is formed from P4, P5, P6 and second from P3, P7, P8 and P9. Group-I intron have open reading frames inserted in loop regions. In group-I intron splicing lariat is not formed. A linear intron is formed.

Mechanism :

Group I introns splicing reaction carried out by two sequential ester-transfer reactions. At P7exogenous guanosine or guanosine nucleotide  docking site is present.

First splicing reaction start with the docking of exogenous guanosine onto the active guanosine-nucleotide site located in P7. After that phosphodiester bond at the 5' splice site located in P1 get attacked by aligned 3'-OH of exogenous guanosine. Due to this reaction 3'-OH group at the upstream exon get free and 5’ end of intron occupied by exogenous guanosine.

Now second ester-transfer reaction takes place in which phosphodiester bond at the 3' splice site located in P10 get attack by aligned 3'-OH group of the upstream exon in P1, leading to release of the catalytic intron and ligation of the adjacent upstream and downstream exons.

Group I introns present in (1) mitochondrial and chloroplast genomes of lower eukaryotes. (2) rRNA, mRNA and tRNA genes in bacterial genomes. (3) rRNA genes in the nuclear genome of lower eukaryotes. (4) In higher plants few tRNA and mRNA genes of the chloroplasts and mitochondria, (5) T4 and T7 bacteriophages.

4.4.2.     Group-II intron splicing :

In group II splicing intron exist in a  large class of self-catalytic ribozymes comprising six structural domains (usually designated I to VI or D1 to D6) and length up to 3 kb. Exogenous GTP not require in group II intron splicing and splicing occur through lariat formation same as major splicing. Six structural domain present in these ribozymes. Some group II intron also encode essential splicing protein.

It assume that splicing of group II intron occur by spliceosome formation as in major splicing. Splicing require Mg2+ ion. In vitro, high salt condition required for Ribozyme activity (e.g., self-splicing). Group II catalytic introns present in rRNA, tRNA, and mRNA of organelles (chloroplasts and mitochondria) in fungi, plants, and protists, and also in mRNA in bacteria.

For processing of phosphoryl transfer reactions group I and II intron use two-metal-ion mechanism same as used by protein polymerases and phosphatases proven by a recently resolved high-resolution structure of the Azoarcus group I intron, thus both known as matalloenzyme.

4.4.3.     Group III intron :

Group III auto splicing are rich in A U and ranging from 95 to 110 nucleotides. Group III introns are shorter than group I and II intron but show similarity with group II introns, having conserved sequences proximal to the splicing site specially 5’ splice site. Splicing occurs through lariat and circular RNA formation and identified in chloroplasts of euglenoid protists mRNA genes.

4.5.         Homing and Retrohoming process :

In homing and retrohoming mechanism an intron is insert a within an intron less cognate sequence, which consist of a particular specific site. Some intron of group I and group II has ORF. These ORF translated in enzyme maturase, endonuclease and reverse transcriptase.

Homing intron posses two enzyme maturase and endonuclease. Maturase enzyme help in splicing while endonuclease responsible for homing.  

A specific restriction site present on DNA sequence gene which not possess homing intron, but this DNA sequence (restriction site) is present only in copy of target gene. In a particular cell, two copies must be present for homing. Thus intron coding endonuclease makes introns as mobile element.

In retrohoming intron, maturase, endonuclease and reverse transcriptase genes are found. Endonuclease cut the intron by double strand break and reverse transcriptase make DNA copy of this mRNA, and thus insert this DNA copy into targeted gene, thus move through RNA arbitrated (mediated) mechanism.

4.6.         Twintrons :

Introns-within-introns called twintrons. This discovered in Euglena chloroplast genome as a group II intron within another group II intron and excised by sequential splicing reactions by lariat intermediates with in  internal and external introns.

4.7.         Alternative Splicing:

Alternative splicing is a process in which exons are joined together in differential method and make different polypeptide from a single mRNA transcript. In alternative splicing introns can be retained or exons can be skipped or extended.

One of the best example of alternative splicing is sex determination in Drosophila. In Drosophila sex of fly is determine by ratio of X-chromosome to autosomes. Fly with X/A ration = 1 develope into female and If ratio of X/A is 0.5 develop into male. There are three gene have an important role to transmit this information to other cell in turn upto the genes whose product involve in development of male and female characterstic. They are sxl (sex lethal), tra (transformer) and dsx (double sex). These gene are transcribe seperatly but their splicing is regulated by product of these gene in sequential cascade manner.

Product of sxl gene help in splicling of tra gene and product of tra gene help in splicing of dsx gene. All genes are normal function fly become female. If one of the gene form dysfunction product fly become male.  In this Splicing mechanism sxl product act as repressor and block the 3’splice site thus functional tra protein responsible for female form by the loss of two exon, and this tra protein responsible for the formation of female specific dsx splice mRNA and so female specific dsx protein containing 430 amino acid and in this case tra act as activator.

If sxl protein dysfunctional than male specific tra protein form and in turn male specific dsx protein formed containing 550 amino acid.

Another example of trans splicing from muscle protein of mammals Troponin T. The Troponin T gene transcript contain five exon. When during splicing the fourth exon skipped than spliced mRNA form α troponin T. and when during splicing the third exon skipped than spliced mRNA form β troponin T.

4.8.         Trans-splicing:

In some lower organism like trypanosoma special type of splicing process found in which exon of two different transcripts ligated together. In trypanosomes, a leader sequence present at the 5’end of m-RNA. This leader sequence joined to other m-RNA exon by trans-splicing. ‘Y’ shaped intron exon intermediate is form This Y-shaped intermediate is analogous to lariat. Now leader exon attacks the splice site which is located at branched intron and coding exon. Thus 2 type structure are obtain one is mature mRNA and second is Y-shaped intron.

Trypanosomas cause african sleeping sickness, till date there is no cure of this disease because trypanosomas has ability to change their receptor, here, N terminal sequence where the Ab bind is continuously change due to trans-splicing and the c terminal remain constant. Trans-splicing also found in C. elegans.

4.9.         RNA Editing

RNA editing is a process in which RNA transcript has some different bases from it template DNA bases. That means in RNA editing process either some bases are added after transcription or some bases are removed from transcript. Somewhere instead of addition or deletion, some base replace with another bases.

Thus RNA editing occur by two mechanism

1.            Substitution

2.            Addition or deletion

1.            Substitution editing: Substitution editing occurs due to chemical alteration in individual base. This alteration cause by some enzymes. These enzymes change one nucleotide into another nucleotide.

For example adenosine deaminase acting on RNA (ADARS) converts Adenine (A) to I (inosine) known as A-I editing. I (inosine) behave like Guanosine in DNA and ribosome also translates Inosine as a Guanosine. Cytidinedeaminase convert Cytocine to Uracil known as C- U editing.

Human APO-B gene is best example of substitution editing. This gene expressed in liver cell and intestinal cell of human. APO-B contains 29 exon which are separated by 28 Intron. In 29 exon total 4564 codons are present. At codon 2153 (CAA) code for glutamine amino acid.

In human liver cells, this get transcribed and translated normally and gene product called apolipoprotein B –100 (contain 4563 amino acid) apolipoprotein B 100 work as cholesterol and lipid transport in blood. But in intestinal cell RNA editing occur in transcript of APO-B gene and at 2153 codon cytocinedeaminase convert Cytocine to Uracil. Thus codon CAA changes in UAA which is stop codon. Due to this final protein contains only 2152 amino acid rather than 4563. In intestine this protein known as APO-48 and help in cholesterol absorption from dietary lipids.

Substitution editing also found in tRNA, rRNA of chloroplast and mitochondria of plants, Receptor of neurotransmetter in mammals brain, encoding mRNA also processed through editing process.

Insertion and deletion editing :

Insertion and deletion process requires RNA molecules known as guide RNA (gRNA). gRNA have complementary sequence to editing site within its central domain, which is in between 5’ (having complementary sequence to pre-mRNA)  and 3’ (having poly U tail) end. First step in addition and deletion process requires binding of guide RNA with unedited transcript.

If some mismatch bases are present in transcript, get loop out, endonucleolytic cut makes and remove mismatched bases. Now another enzymes terminal uridylyl transferase (TUT) add uridine in removal site. Editing occur in 5’-3’ direction. All enzymes responsible for cutting, insertion, deletion and ligation present in 20 S editosome. RNA editing by gRNA reported in kinetoplastid protists mitochondrial mRNAs.

Mechanism involve in deletion editing:

  1. U deletion: A nuclease (endonuclease-because only endonuclease break phosphodiester bond) cut RNA from 3' site of U, which to be deleted.
  2. Now uridyl exonuclease remove UMP at the end of the left-hand RNA fragment, base pairing occurs between bases of the hn mRNA and base of gRNA.
  3. RNA ligase join the two fragment of pre mRNA together.

Insertion editing :

  1. First guide RNA bind with mRNA. If guide RNA fail to exact complementary binding than phosphodiester bond break by nuclease and free 3’OH get created.
  2. Now Terminal Uridylyl transferase (TUTase) insert U base in mRNA.
  3. Finally RNA ligase join two fragments.

4.10.      Ribozymes

RNA used as catalysts in living cells, other than their known roles in information storage and as molecular architectural frameworks are called ribozymes. This idea was so profoundly contrary to the central dogma of molecular biology that it resulted in the award of a Nobel Prize to two of the early proponents, Thomas Cech and Sidney Altman. The discovery was twofold – RNA segments that cut themselves out of larger RNAs (self-splicing introns) and a protein-assisted RNA enzyme (ribonuclease P) that cuts the leader sequences off all transfer RNAs throughout the three organismal domains. RNAs with catalytic activity, revealed the extraordinary characteristic of this molecule, and collaborated the idea that RNA was the first informative polymer. The catalytic properties of ribozymes are majorly due to the capacity of RNA molecules to fold into particular structures. The versatile structures of RNA can allow a single RNA sequence to fold in more than one structure providing them with the virtue of multiple functions.

Ribozymes are found in nucleus, mitochondria and chloroplasts of eukaryotes, as well as in some viruses. These are grouped by their chemical type, but regardless of the type, all RNA are associated with metal ions such as potassium (K+) or magnesium (Mg2+), which play essential roles in catalyzing reactions.

Most ribozymes are involved in the processing of RNA. They either serve as “molecular scissors” or as “staplers” that cleave and ligate  molecules of RNA. Although most targets of ribozymes are RNA, evidence suggests that the assembly of amino acids into a protein that occurs during translation is also catalyzed by RNA, meaning the ribosomal RNA is also a ribozyme.

Group II introns are autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays an essential role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns are also responsible for genome evolution. The structure and catalytic mechanism of group II introns have been elucidated through a combination of genetics, chemical biology, biochemistry, and crystallography. A central active site, containing a reactive metal ion, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions for the behavior of RNA machines.

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