Lecture #13: RNA Processing and Measurement

How do RNA strands leave the nucleus?

• Through nuclear pores

• Movement is mediated by specific transport proteins

Transcription and translation in prokaryotes

• Transcription and translation are couples and occur simultaneously in cytoplasm

• Termination of RNA synthesis occurs at a specific site; the intact RNA is then translated into proteins on ribosomes

Transcription and translation in eukaryotes

• Occur in different cellular locations

  • mRNA must be transported from the nucleus to cytoplasm before translation occurs

    • Transport to cytoplasm is also regulated

• Termination is not well understood, transcript undergroes several modifications before exporting cytoplasm for translation

Major types of RNA processing

• Addition of 5’ CAP

• Addition of a POLY A tail

• RNA splicing

How mRNA is capped

Function of 5’ CAP

• enhances transport of mRNA to cytoplasm by transport proteins

• enhances translation of mRNA

• protects mRNA from degradation

  • masks 5’ end from exo and endo ribonucleases

Polyadenylation

• Adds 50-250 AMP residues added to 3’ end of transcript

• role:

  • Protect RNA from degradation

  • Enhances translation of mRNA

  • Enhances transport of mRNA to cytoplasm by being recognized by specific transport proteins

  • helps identify RNAs for degradation

    • RNAs with short polyA tails are more susceptible to RNAses

Steps in polyadenylation

Splicing

Splice introns from primary transcript

Recognized sequences for splicing

• GU and AG are recognized by the spliceosome

• A middle “A”: Lariat branch point

Lariat model of splicing

• 2’ OH of A (Lariat branch point) attacks phosphodiester bond at 5’ splice site (nucleophilic attack)

• Lariat forms by 5’ (g) to 2’ (a)

• Free OH on exon 1 attacks phosphodiester bond between intron and exon 2 to cleave 3’ junction

• Ligation of exon 1 and exon 2 by formation of a phosphodiester bond→cut is called a lariat which is degraded

  Spliceosome is responsible for this process and holds everything together

Spliceosome

• RNA protein complex with around 70 proteins

  • snRNPs=Small Nuclear Ribonuclear Proteins

  • snRNA + Protein =sn RNPs

  • 5 different snurps (U1,2,4,5,6; 3 is used in ribosomal processing)

• Steps:

• 1 recognizes the 5’ splice site first

• U1 is replaced by U6

• U2 recognizes the branchpoint and protein U2AF (associated factor) recognize the 3’ splice site

  • A at branch site bulges out and doesnt participate in base pair between snurp and pre-mature RNA

• U5 binds to 5’ and 3’ splice sites after initial recognition

• 

Advantages of RNA splicing

• Increases # of proteins made form a single gene by alternative splicing (multiple proteins from a single gene)

• Regulates gene expression

• Introns in DNA bind some transcription factors

What RNAs are effectively transported out of the nucleus?

• Ones that are polyadenylated, spliced, and capped

Methods for measuring transcription

Northern Blots

• measures RNA abundance

  • Related to the rate of synthesis versus the rate of degradation

• Even if transcription rate is the same, the degradation make be different, leading to differences in Northern blot

Run-on assays

• measures transcription rate (mostly initiation rate)

  • DNA is isolate

  • Incubated with a labelled mRNA

  • Hybridize labelled mRNA is spotted with DNA

  • The darkness of the color correlated to the transcription rate

    • each circle is a gene of interest

cDNA

• Complementary DNA

• Solution to not being able to clone RNA

  • mRNA can be converted into DNA using reverse transcriptase

    • RTs: from retrovirus that use RTs to convert their RNA genomes to DNA

Uses of cDNA

• express intron-bearing eukaryotic genes in bacteria

• compare cDNA and genomic DNA to map introns

• measure levels of gene expression

Making cDNA from eukaryotes

1. Separate mRNA and beads by lowing salt or heat

2. Convert RNA into DNA using reverse transcriptase and a primer such as oligo-dT

   1. anneal primer

   2. incubate with reverse transcriptase

      1. Reads 3→5 and synthesizes

3. Add a poly G tail (using terminal transferase)

4. Remove RNA with RNAse H (specific to DNA/RNA hybrids)

5. Make dsDNA by adding C primer, then DNA polymerase

6. Ligate cDNA to linkers containing restriction sites

7. Digest linkers with restriction enzyme

8. Ligate to a vector and transform E. coli

Application of cDNA

1. Make libraries

2. Compare cDNA versus genomic DNA to define structure of the gene and locations of intron

3. Produce a protein from an animal gene in bacteria (bacteria cant splice introns