Preservation of mRNAs in Torpor for Brown Adipose Tissue Activity During Arousal

JORDAN SPALDINGBIOLOGY 3939UNIVERSITY OF COLORADO DENVERAIMEE BERNARD, PhD

Ictidomys tridecemlineatus

Spalding Jordan, Grabek Katie, Martin Sandy

Main points

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

13 Lined Ground Squirrels(Ictidomys tridecemlineatus)

[1] Squirrels and Hibernation*

HibernationHeterothermy in Winter

Non-Shivering ThermogenesisTorpor/Arousal Cycles

Hindle, A. (2013) Martinlab.

[iv]

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

Transcriptome Analysis[2] Increase in Abundance of Transcripts*

Grabek, K. (2013). MartinLab

InterBout Arousal Late Torpor Early Arousal Spring Warm

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5Re

lativ

e Fo

ld C

hang

e

InterBout Arousal Late Torpor Early Arousal Spring Warm0

5

10

15

20

25

30

35

40

Bo

ty T

emp

. ºC

How does transcript abundance increase in torpor?

Enriched for BAT activityCluster Enrichment

ScoreNo. of Genes

Mitochondrion4.04 27

Neutral Lipid Biosynthetic Process 2.74 4

Lipid Catabolic Process2.33 11

Adipocytokine Signaling Pathway 2.17 9

Glycerolipid Metabolic Process 2.14 12

Generation Of Precursor Metabolites And Energy

2.08 13

Tricarboxylic Acid Cycle2.03 5

Lipid Droplet1.93 3

Fatty Acid Metabolism1.38 7

Grabek, K. (2013). MartinLabFurness, D. (2013) Keele University

Stabilized

Transcription in torpor

How can transcript abundance increase?[3] Is it transcription or preservation?*

The experimental approachTotal 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

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

qPCR

Lipe I8B cDNA

Results

GSR LIPE QRSL1 BTG2 Snora

STAP2 CIDEC GAPDH Scarna OGDH

Transcripts with long Poly(A) tails are stable during torporTranscripts lacking/with short poly(A) tails decrease in torpor

Polyadenylation confers stability for select transcripts

Lipe STAP2Snora44 scaRNA 9

IBA LT E-Ar SpW0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

STAP2Percent of Total RNA

Signal transduction

InterBout Arousal

Late Torpor Early Arousal Spring Warm0

10

20

30

40

Bo

ty T

emp

. ºC

InterBout Arousal

Late Torpor Early Arousal Spring Warm0

10

20

30

40

Bo

ty T

emp

. ºC

Total RNA

Long

Actual Abundance

Long

IBA LT E-Ar SpW0

5

10

15

20

25

30

35

Perc

ent o

f Tot

al R

NA

(rec

over

ed)

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

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.

Bibliographyi. 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.