The Transcriptome: a primer on Transcription/RNA

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The Transcriptome a primer on Transcription/RNA

• 2-4-08

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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

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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

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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

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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

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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 which mature mRNAs are read
  to generate polypeptides
• occurs in the cytoplasm.
• In a eukaryotic cell
• The nuclear envelope separates transcription from
  translation

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Molecular Components of Transcription

• Transcription
• Is the DNA-directed synthesis 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 substitutes for thymine

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RNA Polymerase Binding and Initiation of
Transcription

• Promoters
• Signal the initiation of RNA synthesis
• Transcription factors
• Help eukaryotic RNA polymerase recognize promoter
  sequences

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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

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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

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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
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Splicing from pre-mRNA to mature mRNA

• RNA splicing
• Removes intronic sequences
• Joins together exons
• Generates a shorter transcript

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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

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Structure of a Mature mRNA
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RNA Processing A Summary
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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

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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

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Expression Profiling Microarray Analysis

• Gene expression profiling (microarray analysis)
• has enabled the measurement of thousands of
  genes in a single RNA sample

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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

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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 addl ones that I dont have space
  to mentionpoint is, RNA has a broad spectrum
  of structures/functions!)

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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

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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)

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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)

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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.