Year 3 Pharm.D Dr. V. Chitra

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1 Year 3 Pharm.D Dr. V. Chitra

2 RNA processing CENTRAL DOGMA DNA RNA PROTEIN DNA is the genetic material, located in the nucleus. mrna carries a faithful copy of a gene into the cytoplasm for protein synthesis. Proteins are enzymatic and structural elements, made in the cytoplasm.

3 RNA processing DNA RNA discovery of reverse transcriptase (1970) RNA can be back-transcribed into DNA PROTEIN DNA pre-mrna MATURE RNA discovery of pre-mrna splicing (1977) PROTEIN

4 RNA processing DNA PRECURSOR RNAs MATURE RNA PROTEIN Cleavage Nucleotide addition Nucleotide insertion Nucleotide removal Sequence addition Sequence removal Base modification Sugar modification pre-rrna pre-trna pre-mrna Other RNA-related factors affecting expression abundance (combination of transcription and degradation) localization recruitment to ribosomes

5 RNAs that function in RNA processing rrna snornas trna RNase P snornas mrna snrnas grnas mirnas sirnas form complexes with protein, direct nt modifications has both RNA and protein components form complexes with protein, direct nt modifications U1,2,4,5,6 form spliceosomes with many proteins provide sequence information for RNA editing important for regulating gene expression important for regulating gene expression sno, small nucleolar sn, small nuclear

6 RNAs that function in RNA processing RNA functions in RNA processing Site of action typically directed by complementary basepairing Action often catalyzed by associated protein(s) HOWEVER some RNAs ribozymes have catalytic activity self-splicing intron in Tetrahymena rrna hammerhead ribozymes self-cleave snrnas appear to have catalytic activity in pre-mrna splicing

7 rrna processing Cleavage: Pre-rRNA is cleaved to 18S, 5.8S, 28S rrnas; cleavage order is precise (within species). DNA RNA 18S 5.8S 28S ETS ITS1 ITS2 ETS 18S 5.8S 28S ETS ITS1 ITS2 ETS 18S 5.8S 28S ETS ITS1 ITS2 ETS 18S 5.8S 28S ETS ITS1 ITS2 ETS 18S 5.8S 28S Schematic is ETS ITS1 ITS2 ETS generic and not to scale;

8 trna processing Removal of 5 leader and 3 trailer; order not absolute CCA may be encoded (prok.) or added post-transcriptionally (euk.) Acceptor stem sometimes edited Some trnas have introns in the anticodon loop editing intron Many nucleotide modifications

9 rrna processing in nucleolus Modification: both bases and sugars are subject to modification rrnas ~100 riboses are 2 O-methylated 10 bases methylated 95 Us isomerized to pseudous (ψs) modifications occur prior to assembly into ribosomes trnas ~100 kinds of modified nucleotides some incorporated during transcription some chemically modified post-transcription snornas direct many modifications

10 RNA modification snornas modify rrnas, trnas, mirnas, sirnas, and mrnas number variable between organisms; more being found size range ~60 to ~300 nt encoded individually, in polycistronic clusters, or in introns Most C/D snornas (and snrnas) have a 5 trimethylguanosine (TMG) cap. Patients with motor neuron degeneration diseases often develop antibodies that recognize TMG caps. H/ACA snornas direct pseudouridylation C/D snornas direct methylation

11 mrna processing Capping Splicing Polyadenylation Editing Export Localization Translation Turnover From birth to death, an mrna associates with a variety of proteins and other RNAs that modify it directly or affect its abundance and recruitment to ribosomes. Aguilera 2005 mrnp (messenger ribonucleoprotein particle): mrna + associated proteins

12 mrna processing - capping 5 capping required for translation of eukaryotic mrnas mediates initial ribosome binding 7-methylguanosine cap added as RNA exits RNApol II...G linked via a 5-5 pyrophosphate bridge to first nt of mrna...g methylated post-addition...first bases in mrna may also become methylated pause after capping, then cap-binding complex (CBC) associates with cap and elongation proceeds Aguilar 2005

13 RNA processing - splicing Removes blocks of non-coding sequence (introns: intervening sequences), ligates the surrounding coding sequences (exons: expressed sequences). Catalyzed by an RNA/protein complex, the spliceosome, which is composed of five ribonucleoprotein complexes (snrnps). Each snrnp has a distinct small nuclear RNA (snrna): U1, U2, U4, U5, or U6. Each also has a set of common core proteins and some that are specific to the particular snrna. Occurs by two transesterification reactions (no energy required) cis-splicing: both exons on same RNA trans-splicing: exons on different RNAs

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