RNA processing complete.pptx

RNA PROCESSING
Types of RNA
Transcription of pre-rRNA
Modification of pre-rRNA
Trimming and cleavage
Splicing of pre-rRNA
Capping of pre-mRNA

1. RNA PROCESSING PRESENTED BY: PRASHANT VC DEPT OF ZOOLOGY GUK

2. Introduction RNA : ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding,decoding,Aregulation and expression of genes. Types of RNA : ribosomal RNA (rRNA), transport RNA (tRNA) , messenger RNA (mRNA). Each of these RNA are transcribed and futher processed to become fully functional in order to bring out certain biological functions Among them only mRNA goes under translation and remaining tRNA and rRNA along with ribosomal proteins and amino acids carry out the translation process. After transcription all RNAs are in pre-RNA form and by the processing they are converted into mature RNAs.

3. rRNA processing Transcription of pre-rRNA: rRNA is transcribed from rDNA (ribosomal DNA) in nucleolus. There are 4 types of rRNA found in eukaryotic cells 1. 5S rRNA 2. 5.8S rRNA 3. 28S rRNA 4. 18S rRNA. Ribosomes are made up of 2 subunits : 60S (composed of 5S , 28S and 5.8S rRNA) and 40S (18S rRNA and 50 different proteins). Modification of pre-rRNA: This can be classified as different process occurs in different part of nucleolus. The early modification takes place on the dense fibrillar centre of nucleolus which includes pseudouridation of adenine. Late modification takes place in granular components of nucleolus and it involves methylation of nucleotides.

4. Trimming and clevage: A long stretch of RNA is trimmed into two rRNAs : 1. 5S rRNA 2. 47S rRNA by endonuclease and exonucleases. 47S rRNA is precursor of 28S , 5.8S and 18S rRNA. Synthesis of 5S rRNA is done by enzyme RNA polymerase III and other rRNAs are synthesized by RNA polymerase I.

5. Splicing of pre-rRNA: the pre-rRNA sequence do contain some introns between to exons therefore needs splicing. The sequence contains a conserved nucleotide adenine (A) on which the 2` hydroxyl oxygen carrying lone pair of electron attacks the phosphate of 1 st exon-intron junction acting as an electrophile. And the hydroxyl group becomes free on the junction which attacks the phosphate group of 2 nd exon-intron junction removing a lariant. The two exons are joined together.

6. tRNA processing Transcription of pre-tRNA: tRNA genes are transcribed and processed in the nucleus but for further charging they are sent to cytoplasm. From tRNA gene primary transcript is produced and different transcripts are joined to make one pre-tRNA. There are 21 different tRNAs for 21 different amino acids. Clevage and trimming: 5` end (5` leader sequence) and 3` end seq of the pre-tRNA is cleaved by endonuclease enzyme further trimmed by exonuclease enzyme. These enzymes are Rnase p and Rnase E/F fir 5` end and Rnase D for 3` end.

7. Addition of CCA: At the 3` end of pre-tRNA a sequence of cytosine-cytosine-adenine is added by the enzyme tRNA nucleotidyl transferase. The addition is called quality control. Amino acids are loaded at the site of addition. Amino acids are added on 3` terminal of CCA by aminoacyl tRNA synthetases to form aminoacyl tRNA. Modification of nucleotides: On average atleast 12 nucleotides are modified in each pre-rRNA. Examples : Adenine is converted to pseudouridine. Uridine is converted to dihydrouridine. Adenine is converted to inosine.

8. Splicing: The pre-tRNA molecule contains introns which is to be removed out. The removal of the intron is performed by endonucleases such as Sen34,Sen54, Sen2 and Sen15. On removal of intron one 2`3`cyclic phosphate group and 5`OH group is exposed and are further joined with the help of Phosphodiesterase,ligaes,kinase and phosphatase. On removal of intron pre-tRNA is converted into tRNA. Charging of tRNA: The charging of tRNA is actually addition of amino acids in the 3’ end off tRNA. This process is done in the cytoplasm by aminoacyl tRNA synthetases.

9. mRNA processing Transcription of pre-mRNA: Pre-mRNA is transcribed and processed in nucleus and after formation of mature mRNA it is sent to cytoplasm. Processing of Pre-mRNA basically involves three major processes: Capping : addition of 7M guanine nucleotide on 5` terminal. Polyadenylation: addition of polyadenine tail on the 3` terminal. Splicing : introns are removed and exons are joined. After capping polyadenylation and splicing of pre-mRNA it is called as matured mRNA. Mature mRNA is sent to cytoplasm fir translation.

10. Capping of pre-mRNA Capping of pre-mRNA means addition of 7me guanine nucleotide on the 5`terminal of pre-mRNA. Capping starts as the transcript of mRNA emerges after transcription. Uncapped pre-mRNA possesses triphophaste group in the terminal nucleotide which is to be capped (5`pppNpN n 3`). Triphosphatase enzyme removes one phosphate group leaving two on the 5` terminal nucleotide. (5`ppNpN n 3`). By the action of guanylyl transferase enzyme and GTP , guanine with one phosphate group is added on the 5` terminal. (5`GpppNpN n 3`). The guanine added in the 5` terminal is methylated on the 7 th nitrogen by mRNA guanine-N7-methione transferase. (5` 7me GpppNpN n 3`). In some cases further nucleotides are also methylated called as 2 nd cap and 3 rd cap.

11. The CEC capping enzyme complex binds to polymerase II enzyme as soon as the transcript emerges by transcription to bring out cappong process. Importance of capping : Regulation of nuclear export. Prevention from degradation by exonucleases. Promotion of translation. Promotion of 5` proximal intron excision.

12. Polyadenylation Polyadenylation is the process of addition of multiple adenine nucleotides on the 3` terminal of pre-mRNA. This process starts as soon as the termination of transcription is done. Just before some nucleotides at 3`end there is a cleavage sequence present AAUAA which is recognised by CPSF ( cleavage and polyadenylation signal factor) and CSF (Cleavage stimulating factor). These proteins binds toh the cleavage site and attract nuclease enzyme to cleave the stretch of nucleotide after the sequence. On the free 3` end of pre-mRNA polyA polymerase enzyme adds adenine nucleotides. A protein called polyA-binding protein ensures addition of enough adenine nucleotides.

13. In some genes the ckeavage proteins add a poly A tail at one of the several cleavage sites. Therefore, polyadenylation can produce more than one transcripts (alternate polyadenylation)to form a singke gene similar to alternate splicing. Importance of polyadenylation: Polyadenylation provides stability to mRNA Prevent degradation by exonucleases Exonucleases degradation the terminal nucleotides,to compensate muktiple adenines are added so that if exonuclease degrades the nucleotides coding sequences are saved.

14. 5`cap Polymerase II AAUAA DNA Cleavage signal recognised by CPSF and CSF Nuclease activity Poly A polymerase and polyA binding protein (PAB) AAAAAAAAAAAAAAAAAAAA 3` 5` 5` 5` 3` 3` PAB PAB PAB Pre-mRNA Pre-mRNA Pre-mRNA Pre-mRNA CSF CPSF

15. Splicing There are several sequences on a pre-mRNA. Most introns starts with a sequence of GU (splice donor site) and ends with AG (splice acceptor site). The presence of only these two sequences cannot confirm the presence of mRNA. A conserved Adenine is always present 20-50 basepairs upstream of acceptor site. A is followed by a polypyrimidine tract. The consensus sequence of branch site is CU(A/G) A (C/U) where A is conserved. 5’splice site: the exon-intron boundary at the 5’ end of the intron 3’ splice site: the exon-intron boundary at the 3’ end of the intron Branch point site: an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).

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17. The splicing is mediated by a group of proteins called snRNPs (Small nuclear ribonucleo proteins). U1 U2 U4 U5 U6. This process can follow two mechanisms Self splicing/ cis-splicing: Splicing in single RNA. Lariat shape Common Trans splicing: Two different RNAs Y shape Rare ( c. Elegans and higher eukaryotes) Spliceosome mediated Splicing

18. Spliceosome is a group of snRNPs (small nuclear ribonucleo proteins). U1 binds on the 1 st exon-intron junction. A protein called BBP (branch site binding protein) binds to the branch site ( A ) and U2AF ( U2 auxiliary factor) which helps the U2 snRNP to bind with branch site binds with the BBP. A complex of U4,U5,U6 binds the the stretch of intron and fold them to form a lariat. After the cleave of splice site these proteins terminate lariat from the mRNA. And exons are joined to form a matire mRNA.

19. Cis splicing / Self splicing This process takes 2 steps : Step 1 : The OH group of Conserved A carrying lone pair of electron and acts as electrophile and attacks the phisphoryl group of G in 5` end of intron. 5` exons is released. 5` end of intron makes a three way junction. Step 2 : The OH group of 5` exon attacks the 3` splice site of intron. 5`exon and 3`exon are joined and a lariat intron is removed Tbis process happens when there is one RNA to be spliced.

20. Trans splicing Trans splicing is done when there are 2 or more RNAs to be spliced together.

21. References Snustad, D. Peter, and Michael J. Simmons.  Principles of genetics . John Wiley & Sons, 2015. Allison, Lizabeth A.  Fundamental molecular biology . Blackwell Pub., 2007. Albert, Bruce. "Molecular biology of the cell." (2008). https://courses.lumenlearning.com/boundless-biology/chapter/rna-processing-in-eukaryotes https://reactome.org/content/detail/R-HSA-72312

22. Thank-you