Transcript No Slide Title

CMS
A long noncoding RNA regulates photoperiod-sensitive male sterility,
an essential component of hybrid rice(2012) doi:
10.1073/pnas.1121374109
A non-coding RNA locus mediates environment-conditioned male
sterility in rice. (2012) Cell Research 22:791–792.
doi:10.1038/cr.2012.43
Comparative expression profiling of miRNA during anther
development in genetic male sterile and wild type cotton. (2013) BMC
Plant Biology 13:66
Differential Proteomic Analysis of Anthers between Cytoplasmic
Male Sterile and Maintainer Lines in Capsicum annuum L.(2013) Int.
J. Mol. Sci. 14(11), 22982-22996; doi:10.3390/ijms141122982
Transcriptome map of plant mitochondria reveals islands of
unexpected transcribed regions (2011) BMC Genomics 12: 279.
Heterozygous alleles restore male fertility to cytoplasmic male-sterile
radish (Raphanus sativus L.): a case of overdominance(2013) J. Exp.
Bot. 64: 2041-2048.
Aging
Genome
1. DNA damage
2. Epigenetic shifts
3. Telomere shortening
Cellular level
1. Mitochondria: ROS, DNA damage, other
2. Misfolded proteins
3. Dysfunctional stem cells
Organismal level
1. Autoimmune, other defects in immune system
2. Defective signaling
Journal Clubs
Spend ~ 5’ setting the stage:
• what is the general question?
• Why is it important?
• What was previously known?
• What were the outstanding questions?
Journal Clubs
Spend ~ 5’ setting the stage:
• what is the general question?
• Why is it important?
• What was previously known?
• What were the outstanding questions?
Then state the specific question addressed in your paper
Journal Clubs
Then state the specific question addressed in your paper
Next explain how they studied it
• General overview of techniques first, then specifics
• What were they trying to do?
• how did they do it?
Journal Clubs
Then state the specific question addressed in your paper
Next explain how they studied it
• General overview of techniques first, then specifics
• What were they trying to do?
• how did they do it?
Then describe their results
Journal Clubs
Then describe their results
• General overview first
• Then specific experiments
Journal Clubs
Describe their results
• General overview first
• Then specific experiments
• Specific purpose of each experiment
• How they tested it
• Data they collected
• Controls!!
• How they analyzed it
Journal Clubs
Describe their results
• General overview first
• Then specific experiments
• Specific purpose of each experiment
• How they tested it
• Data they collected
• Controls!!
• How they analyzed it
Conclusions they drew
Journal Clubs
Describe their results
• General overview first
• Then specific experiments
• Specific purpose of each experiment
• How they tested it
• Data they collected
• Controls!!
• How they analyzed it
Conclusions they drew
Your interpretation
• Do you agree?
• How could they improve?
Non-coding RNA
Nearly ½ of human genome is transcribed, only 1% is
coding
• 98% of RNA made is non-coding
Non-coding RNA
Nearly ½ of human genome is transcribed, only 1% is coding
• 98% of RNA made is non-coding
•Fraction increases with organism’s complexity
Non-coding RNA
Structural
1. rRNA
2. tRNA
3. snRNA
4. snoRNA
5. cleavage: RNAses P & MRP, U3, snR30, etc
Regulatory
1. Small
• siRNA
• miRNA
2. Long
• Activator
• Enhancer
• silencing
Other ncRNA?
Incredible diversity of
functions!
• Epigenetic
• Directly regulating
transcription
• Post-transcriptional
regulation
Some are made by Pol
II, others by Pol III
HID1 ncRNA
1. Extracted total RNA from a mix of tissues, separated by
size and sequenced 50-300 nt fraction
HID1 ncRNA
1. Extracted total RNA from a mix of tissues, separated by
size and sequenced 50-300 nt fraction
2. BLAST to find relatives (and discard tRNA, etc)
HID1 ncRNA
1. Extracted total RNA from a mix of tissues, separated by
size and sequenced 50-300 nt fraction
2. BLAST to find relatives (and discard tRNA, etc)
3. T-DNA express to find mutants
HID1 ncRNA
1. Extracted total RNA from a mix of tissues, separated by
size and sequenced 50-300 nt fraction
2. BLAST to find relatives (and discard tRNA, etc)
3. T-DNA express to find mutants
4. ordered seeds for ones they found interesting
HID1 ncRNA
• ordered seeds for ones they found interesting
• Grew them under varying light conditions
•
•
•
HID1 ncRNA
ordered seeds for ones they found interesting
Grew them under varying light conditions
Found one specifically defective in sensing continuous red
•
•
•
HID1 ncRNA
ordered seeds for ones they found interesting
Grew them under varying light conditions
Found one specifically defective in sensing continuous red
HID1 ncRNA
• Found one specifically defective in sensing continuous red
• Confirmed that the HID1 ncRNA was responsible by adding back
wt gene to “rescue phenotype”
•
•
•
•
HID1 ncRNA
Confirmed that the HID1 ncRNA was responsible by adding back
wt gene to “rescue phenotype”
Checked expression
Constitutive
Not regulated by light
HID1 ncRNA
•
•
Checked expression
• Constitutive
• Not regulated by light
Tested sequence requirements to rule out ORFs
•
•
HID1 ncRNA
Tested sequence requirements to rule out ORFs
Tested structural requirements under cR
•
•
HID1 ncRNA
Tested sequence requirements to rule out ORFs
Tested structural requirements under cR
• Both SL2 and SL4 are needed for it to function
•
•
•
HID1 ncRNA
Tested sequence requirements to rule out ORFs
Tested structural requirements under cR
Searched for targets by RNA-seq
•
•
HID1 ncRNA
Searched for targets by RNA-seq
HID1 represses PIF3 expression under cR
HID1 ncRNA
• Does HID1 act alone?
• Added an S1 aptamer to HID1 RNA to help find it
HID1 ncRNA
• Does HID1 act alone?
• Added an S1 aptamer to HID1 RNA to help find it
• Was part of an ~ 500 kDa complex
HID1 ncRNA
•
Does HID1 act alone?
• Added an S1 aptamer to HID1 RNA to help find it
• Was part of an ~ 500 kDa complex
• ChIP-PCR showed that the complex bound the PIF3 promoter
HID1 ncRNA
•
Does HID1 act alone?
• Added an S1 aptamer to HID1 RNA to help find it
• Was part of an ~ 500 kDa complex
• ChIP-PCR showed that the complex bound the PIF3 promoter
• Is HID1 conserved in evolution?
HID1 ncRNA
•
Does HID1 act alone?
• Added an S1 aptamer to HID1 RNA to help find it
• Was part of an ~ 500 kDa complex
• ChIP-PCR showed that the complex bound the PIF3 promoter
• Is HID1 conserved in evolution?
• Are the orthologs functional?
HID1 ncRNA
•
Does HID1 act alone?
• Added an S1 aptamer to HID1 RNA to help find it
• Was part of an ~ 500 kDa complex
• ChIP-PCR showed that the complex bound the PIF3 promoter
• Is HID1 conserved in evolution?
• Are the orthologs functional?
• What next?
Mitochondria and the immune system
• Mito play important role in recognizing & fighting viruses
• Via RLR (retinoic acid-inducible receptors) pathway that detects
dsRNA
• MAVS (mitochondrial antiviral
signaling) protein on MOM is key
• dsRNA receptors bind MAVS &
trigger interferon & cytokine
synthesis
Programmed cell death vs necrosis
Necrosis:
progressive loss of membrane integrity
swelling of cytoplasm, release of cell constituents
Often follows irreversible injury
Passive
Indiscriminate
PCD
Active
Orderly process mediated by
intracellular death programs
May or may not be due to an
external factor
Nuclear condensation
Condensation of PM
Apoptosis
Ordered process that breaks cell into easily recycled pieces
Caspases digest proteins
CAD digests DNA
Apoptosis
Two basic steps: commitment and execution
Commitment depends on interplay between various signals
Bax & Bcl2 have opposite effects
2 main pathways: extrinsic & intrinsic
Apoptosis
2 main pathways: extrinsic & intrinsic
Tumor necrosis factor and Fas ligand = extrinsic signals that can
trigger apoptosis via death receptor
Apoptosis
2 main pathways: extrinsic & intrinsic
Tumor necrosis factor and Fas ligand = extrinsic signals that can
trigger apoptosis via death receptor
Bind receptors in PM (TNFR or fas)
Tumor necrosis factor and Fas ligand = extrinsic signals that can
trigger apoptosis via death receptor
Bind receptors in PM (TNFR or fas)
Receptors activate FADD & TRADD: Adaptors with death domains
that bind receptor’s DDs
Receptors activate FADD & TRADD: Adaptors with death domains
that bind receptor’s DDs
Procaspase 8 binds FADD
Receptors activate FADD & TRADD: Adaptors with death domains
that bind receptor’s DDs
Procaspase 8 binds FADD
Procaspase 8 is processed
to caspase 8
= initiator caspase
Procaspase 8 binds FADD
Procaspase 8 is processed
to caspase 8
= initiator caspase
Caspase 8 converts procaspase 3
to active form = executioner
Procaspase 8 binds FADD
Procaspase 8 is processed to caspase 8
= initiator caspase
Caspase 8 converts procaspase 3
to active form = executioner
Caspase-3 & CAD
execute the cell