OBJ - RNA Metabolism Flashcards

1
Q

Understand the basic mechanism of pre-mRNA splicing, including the sequences and information that define the splice sites.

A
  • nascent pre-messenger RNA pre-mRNA
  • removal of introns & joining of exons
  • catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs)

Mechanism = Double Transesterification

  • Hydroxyl on intron attacks nucleophilic P on exon 1 on the 3’ C
  • Exon 1’s 3’ end OH attacks P on exon 2
  • Exon1-P-Exon 2 & Lariat intron
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2
Q

Define alternative splicing and understand how it increases protein diversity.

A
  • Alternative Splicing (~90% of genes)
  • Results in a single gene coding for multiple proteins
  • Increases diversity & variability for 1 genetic code

Ex: Allows 1 cell to secrete & have membrane bound forms of protein with the same variable region of RNA or 2 different peptides

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3
Q

Understand the different mechanisms by which mutations can give rise to genetic diseases.

A

15-50% Splicing Defects

Mutations in splicing enhancers/silencers cause disease not point mutations (often silent)

Cause wrong splice sites for introns/exons; may added or subtracted

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4
Q

Know how the example disease, Spinal Muscular Atrophy, occurs and how understanding of its molecular biology is likely to lead to effective treatments

A

Spinal Muscular Atrophy (SMA) is a genetic, neuromuscular disease characterized by dysfunction and death of motor neurons in the spinal cord, generally leading to death in childhood.

The incidence of SMA is 1 in 10,000 (among the most common genetic causes of infant mortality.)

There is no current treatment. (only palliative care)

SMA is caused by a mutation of the SMN1 gene (SMN = Survival of Motor Neurons).

Both copies of SMN1 must be mutated to generate the disease; SMN 2 cannot substitute for SMN 1

Changes exonic splicing enhancer -> SR protein no longer binds, resulting insufficient recruitment & skips exon 7 -> SMN delta7 (nonfunction, unstable)

Mutation is a silent mutation -> C to T, but still Phe SNM2 not spliced properly

Treatment: try to block hnRNP by blocking ISS with an oligonucleotid; allows SR protein to recruit U1 & U2 (splicing factors) -> corrects successfully in lab & with mice

**SMN required for assembly of snRNPs (U1-U6)

-still unknown why defect in SMN causes SMA & is seen in nerve cells

4 total alleles = 2 SMN 1’s & 2 SMN 2’s

  • SMN1 = dominant; so need both to be nonfunction so SMN2’s defect is expressed/manifested
  • SMN2 = defective copy of SMN 1
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5
Q

Spilcing Defects

A

Large target size with intronic/Exonic splicing enhancers/silencers

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6
Q

Spliceosome

A

snRNPs = small nuclear Ribonucleoprotein particles
- U1-U6
U2 & U6 = catalytically active protion
followed by 2 transesterifications -> spliced exons & lariat intron

(Exon 1/upstream exon = AG)/GUAAGU
NCAG/(G = Exon 2/downstream exon )

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7
Q

Branch Site (intron)

A

they contain a branch point, a particular nucleotide sequence near the 3’ end of the intron that becomes covalently linked to the 5’ end of the intron during the splicing process, generating a branched (lariat) intron

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8
Q

Splicing Enhancers/silencers

A

Exonic/Intronic Splicing Enhancers:
-binding proteins that recruits U1 & U2 to splice the sites

Exonicx/Intronic Splicing Silencers
- binding proteins that interfere with the ID of the splice site

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9
Q

SR protein

A
  • RECRUITS SPLICING FACTORS
  • RNA binding domain + RS domain (activation domain)
  • bound to ESE/ISE; recruits the splicing machinery -> U1 & U2 snRNPs to initiate splicing
  • each has an RNA binding domain (RRM) that binds to specific RNA sequences & RS domain
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10
Q

hnRNP proteins

A
  • bind to silencers (ISS & ESS)

- blocks SR protein recruiting splicing factors

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