The Transcriptome: a primer on Transcription/RNA

1. The Transcriptome: a primer on Transcription/RNA 2-4-08

2. A review of the ‘omes • The genome • Is all of the genetic information of an organism • Is heritable • Is encoded in DNA • The proteome • Is a collection of proteins present in a certain cell type at any given time • Is composed of amino acids, which are then arranged into complex protein structures • The transcriptome • Is the set of messenger RNA molecules (“transcripts”) produced in a population of cells

3. Why study the transcriptome? • Studying gene expression at the level of RNA • Provides valuable information about the up/down regulation of closely related messages • Enables researchers to determine copy-count and life-expectancy of RNA messages • Can build a view of a specific transcriptional view of a the metabolic state of a cell • Transcriptional profiling • Is used to identify and count individual species of mRNA with a given cell/group of cells • Involves the analysis of thousands of transcripts

4. RNA, cousin of DNA • RNA (ribonucleic acid) • Is produced from the coding regions of a DNA molecule • Is a type of nucleic acid (also found in the nucleus of the cell) • Is single stranded • Contains the sugar ribose (vs deoxyribose in DNA) • Consists 4 different nucleotides: • Guanine, cytosine, adenine, and uracil

5. Relationship between Genes, RNAs and Protein, a Review • In order for a DNA codes to be made into a gene product • It must first be “read” to a generate RNA molecule • The RNA molecule may or may not be the final gene product

6. Flow of Genetic Information within a Cell • Transcription • is the process by which genes are used as a template for mRNA synthesis. • occurs in the nucleus. • Translation • is the process by whichmature mRNAs are “read” to generate polypeptides • occurs in the cytoplasm. • In a eukaryotic cell • The nuclear envelopeseparates transcription from translation

7. Molecular Components of Transcription • Transcription • Is the DNA-directedsynthesis of RNA • RNA synthesis • Is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides • Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutesfor thymine

8. RNA Polymerase Binding and Initiation of Transcription • Promoters • Signal the initiation of RNA synthesis • Transcription factors • Help eukaryotic RNA polymeraserecognizepromoter sequences

9. Elongation of the RNA Strand • As RNA polymerase moves along the DNA • It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

10. Termination of Transcription • Eukaryotic cells • Modify RNA after transcription • Enzymes in the eukaryotic nucleus • Modify pre-mRNA in specific ways before the genetic messages are dispatched to the cytoplasm

11. A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end TRANSCRIPTION DNA Polyadenylation signal Protein-coding segment Pre-mRNA RNA PROCESSING 5 3 mRNA G P P AAA…AAA P AAUAAA Ribosome TRANSLATION Start codon Stop codon Poly-A tail 5 Cap 5 UTR 3 UTR Polypeptide Alteration of mRNA Ends • Each end of a pre-mRNA molecule is modified in a particular way • The 5 end receives a modified nucleotide cap • The 3 end gets a poly-A tail Figure 17.9

12. Intron 5 Exon Exon Intron Exon 3 5 Cap Poly-A tail Pre-mRNA TRANSCRIPTION DNA 30 31 104 105 146 1 Pre-mRNA RNA PROCESSING Introns cut out and exons spliced together Coding segment mRNA Ribosome TRANSLATION 5 Cap Poly-A tail mRNA Polypeptide 1 146 3 UTR 3 UTR Splicing: from pre-mRNA to mature mRNA • RNA splicing • Removesintronic sequences • Joins together exons • Generates a shorter transcript

13. Splicing machinery • Splicing of eukaryotic genes • Requires a protein complex known as the spliceosome • Is dictated by specific nucleotide sequences that flank the intron/exon boundary

14. Structure of a Mature mRNA

15. RNA Processing: A Summary

16. cDNAs • Complementary DNA (cDNA) • Is DNA synthesized from a mature (fully spliced/edited) RNA template • Is obtained using an enzyme called reverse transcriptase • A cDNA library • Is a collection of cDNAs representing all or part of the expressed genes within a cell or population of cells at a given point in time

17. ESTs • Expressed Sequence Tags (ESTs) • Are short sub-sequences of a transcribed, spliced sequence • May be used to identify gene transcripts • Are instrumental in gene discovery and sequence determination • Generating ESTs • Involves sequencing cloned mRNAs (ie cDNAs) • Produces short (500-800 nucleotide) sequences corresponding to portions of expressed genes

18. Expression Profiling: Microarray Analysis • Gene expression profiling (microarray analysis) • has enabled the measurement of thousands of genes in a single RNA sample

19. Gene-expression profiling and medicine • By obtaining samples of cancerous tissue and generating a gene-expression profile • A “molecular signature” of a cancer can be obtained, which could be potentially used for determining treatment options/prognosis

20. Diversity of RNAs • pre-mRNAs • Are the initial messages produced from a DNA template • Will go through various modifications such as splicing and the addition of a cap and tail • mRNAs • Are the mature messages that have been modified from their initial pre-mRNA state • Code for basic cellular processes of the cell • Other species of RNAs include • Regulatory RNAs such as miRNAs, siRNAs and dsRNAs, all of which can inhibit protein production • tRNAs and rRNAs, both of which are involved in translation/protein production • Small nuclear RNAs (snRNPs), which play a structural and catalytic role in the spliceosome (and even some add’l ones that I don’t have space to mention…point is, “RNA” has a broad spectrum of structures/functions!)

21. Formation and Function of RNA Secondary Structure: Hairpins • Hairpins • are a common secondary/ tertiary structure in mature mRNAs • Require complementarity between part of the strand

22. Post-transcriptional Regulatory Mechanisms • Regulatory messages (miRNAs and siRNAs) • Prevent mRNAs from being translated into protein • Are short and lack sequences associated with ribosomal binding (ie the translational machinery)

23. RNAi, an Overview • RNA interference (RNAi) • Is a process in which double-stranded RNA triggers the degradation of a homologous messenger RNA (sharing sequence-specific homology to particular "target" mRNAs)

24. Anti-sense technology to study gene function • By introducing exogenous, dsRNAs that are complimentary to known mRNA's into a cell • It is possible to specifically destroy a particular mRNA, thereby diminishing or abolishing gene expression. • The technique has proven effective in Drosophila, C. elegans, plants, and recently, in mammalian cell cultures.