summary of research completed at Martinlab in 2013.

1. Preservation of mRNAs in Torpor
for Brown Adipose Tissue Activity
During Arousal
JORDAN SPALDING
BIOLOGY 3939
UNIVERSITY OF COLORADO
DENVER
AIMEE BERNARD, PhD
Ictidomys tridecemlineatus
Spalding Jordan, Grabek Katie, Martin Sandy

2. Main points
• Squirrels, hibernation and BAT
• Increase in abundance of transcripts
• Is it transcription or preservation?

3. 13 Lined Ground Squirrels
(Ictidomys tridecemlineatus)
[1] Squirrels and Hibernation*

4. Hibernation
Heterothermy in Winter

5. Non-Shivering Thermogenesis
Torpor/Arousal Cycles
Hindle, A. (2013) Martinlab.

6. [iv]

7. Jastroch et al., Essays Biochem. (2010) 47: 53–67

8. Transcriptome Analysis
[2] Increase in Abundance of Transcripts*
Grabek, K. (2013). MartinLab
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
InterBout Arousal Late Torpor Early Arousal Spring Warm
RelativeFoldChange
0
5
10
15
20
25
30
35
40
InterBout Arousal Late Torpor Early Arousal Spring Warm
BotyTemp.ºC

9. How does transcript abundance
increase in torpor?

10. Enriched for BAT activity
Cluster Enrichment
Score
No. of
Genes
Mitochondrion
4.04 27
Neutral Lipid Biosynthetic
Process 2.74 4
Lipid Catabolic Process
2.33 11
Adipocytokine Signaling
Pathway 2.17 9
Glycerolipid Metabolic
Process 2.14 12
Generation Of Precursor
Metabolites And Energy
2.08 13
Tricarboxylic Acid Cycle
2.03 5
Lipid Droplet
1.93 3
Fatty Acid Metabolism
1.38 7
Grabek, K. (2013). MartinLabFurness, D. (2013) Keele University

11. Stabilized
Transcription
in torpor
How can transcript abundance increase?
[3] Is it transcription or preservation?*

12. The experimental approach
Total RNA
Design Primers
qPCR total RNA
Hybridize Oligo(dT)Make cDNA
Clone
Sequence
Standard Curve
Elute low salt Elute high salt
qPCR long Poly(A) qPCR short poly(A)
Compare + Analyze
Compare to genome

13. Isolation and Sequencing
JH393516.1 137554 to 137652 (+) [LIPE (intron)]
TGGTGTCAAGCAGCCACAAA [LIPE_I8B_FOR primer] (aligned)
AAGTTGGCCGAGCCTCCTGCTGTGGTCCAGGAGACAGCTGGAACAGGGCACCAAGCAT
GGCAATAAAGCCTCATGCTGA [LIPE_I8B_REV primer] (aligned)
GSR LIPE QRSL1 BTG2 Snora
STAP2 CIDEC GAPDH Scarna OGDH

14. qPCR
Lipe I8B cDNA

15. Results
GSR LIPE QRSL1 BTG2 Snora
STAP2 CIDEC GAPDH Scarna OGDH
Transcripts with long Poly(A) tails are stable during torpor
Transcripts lacking/with short poly(A) tails decrease in torpor
Polyadenylation confers stability for select transcripts
Lipe STAP2Snora44 scaRNA 9

16. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
IBA LT E-Ar SpW
STAP2
Percent of Total RNA
Signal transduction
0
10
20
30
40
InterBout Arousal Late Torpor Early Arousal Spring Warm
BotyTemp.ºC
0
10
20
30
40
InterBout Arousal Late Torpor Early Arousal Spring Warm
BotyTemp.ºC
Total RNA
Long
Actual Abundance
Long
0
5
10
15
20
25
30
35
IBA LT E-Ar SpW
PercentofTotalRNA(recovered)

17. Conclusion
• Transcripts ‘increased’ in torpor by stabilization
• Most transcripts degraded in torpor
• Steady state decreases

18. Acknowledgements
• I acknowledge support from the APS’s Integrative
Organismal System Physiology Fellowship
• Thank you to Sandy Martin for being an hands-on
lab PI and persistent in developing my knowledge
• Thank you to Katie Grabek for providing technical
support and walking me through procedures
• Thanks to Allyson Hindle for help with concepts
and troubleshooting
• Thanks to Greg Florant for administrative and
technical support
• Thanks to Vishnu Raman, Ross McNeill, Kelsi
Grogan and Nico Roberts for being there for my
‘stupid’ questions.

19. Bibliography
i. AMREI JÄNICKE, JOHN VANCUYLENBERG, PETER R. BOAG, ANA TRAVEN,
and TRAUDE H. BEILHARZ. ePAT: A simple method to tag adenylated RNA
to measure poly(A)-tail length and other 39 RACE applications. RNA.
2012; (18)6.p1-7.
ii. Da Wei Huang,, Brad T Sherman, & Richard A Lempicki. Systematic and
integrative analysis of large gene lists using DAVID bioinformatics
resources. Nature Protocols. 2009; (4)1.p44-57
iii. Hedda A. Meijer, Martin Bushell, Kirsti Hill, Timothy W. Gant, Anne E.
Willis, Peter Jones and Cornelia H. de Moor. A novel method for poly(A)
fractionation reveals a large population of mRNAs with a short
poly(A)tail in mammalian cells. Nucleic Acids Research. 2007;(35)19.p.1-
13.
iv. Martin, Sandy. (2013) Research Strategy. p.1-17.
v. michael a. frohman, michael k. dush, and gail r. martin. Rapid
production of full-length cDNAs from rare transcripts:
Amplificationusingasinglegene-specificoligonucleotideprimer. Proc.
Nati.Acad. Sci. USA. 1988; (85) p8998-9002.