small RNAs are Short (~18-30 nucleotides) Non-coding molecules. Elucidation of miRNA function depends on the recognition of their target molecules (mRNA transcripts) RNA degradation is a constant reaction in the living cells The total products from RNA decay are uniformly defined as RNA degradome Degradome sequencing is a powerful technique for the detection of cleavage sites of miRNA targets Connects high-throughput Next-Generation Sequencing (NGS) and target predictions for miRNAs Degradome sequencing can provide information about transcripts that undergo degradation by miRNAs 3 case studies Muhammed Ameer Masters Seminar - 2022 Department of Plant Biotechnology Kerala Agricultural University

1. MUHAMMED AMEER
2021-11-133
Department of Plant Biotechnology
Major Advisor: Dr Rehna Augustine
Degradome sequencing and
small RNA targets: What
information it can offer?

2. CONTENTS
• Small RNAs
• RNA degradome
• Degradome sequencing
• Case studies
• Future prospects
• Summary and Conclusion
2

3. The small RNAs (smRNAs)
• Short (~18-30 nucleotides)
• Non-coding molecules
• Act as negative regulators of translation
• Diverse role in cellular and developmental processes
• Major classes of smRNAs:
 microRNA (miRNA)
 small interfering RNA (siRNA)
 piwi-interacting RNA (piRNA)
3

4. The microRNAs
“Challenge to central dogma of biology”
Features
• Consist of ~20-24 nt in plants
• Precursor – stem loop hairpin
• Evolutionarily conserved
• Present in high copy number
Function in plants
• Regulate plant development
• Involved in signal transduction
• Defense response
• Stress responses
Transcribed by RNA polymerase II and possess polyadenylated tails
4

5. • The precursor of miRNA (pri-miRNA)
transcribed in the nucleus
• Processed by Dicer protein to generate the
mature miRNA in cytoplasm
• One strand is incorporated into the RNA-
induced silencing complex (RISC)
• Complementary base pairing directs RISC
to either destroy/block the mRNA
Biogenesis and mode of action
5
(Meltzer, 2005)

6. Why to study miRNAs in plants?
• Basic research ‐ to study regulatory roles in plant growth and
development
• Crop improvement through genetic engineering – miRNAs
regulate key traits
• Development of miRNA based markers
6

7. miRNA target identification
• Elucidation of miRNA function depend on the recognition of
their target molecules (mRNA transcripts)
• Methods to identify miRNA targets and their validation
include:
 Bioinformatics tools
 Quantitative RT-PCR
 Random Amplification of cDNA Ends (RACE)
 Degradome sequencing
7

8. RNA degradome
• RNA degradation is a constant reaction in the living cells
• The total products from RNA decay are uniformly defined
as RNA degradome
• Hence, RNA degradome is a rich deposit of transient
fragments containing abundant information of RNA related
events such as processing and regulation
8

9. Degradome sequencing
• Degradome sequencing is a powerful technique for the
detection of cleavage sites of miRNA targets
• Connects high-throughput Next-Generation Sequencing
(NGS) and target predictions for miRNAs
• Degradome sequencing can provide information about
transcripts that undergo degradation by miRNAs
9

10. • mRNA degradation leads
to specific degradation
products which carry a
phosphate group at the 5′
ends instead of the 5′ mRNA
cap
Principle of degradome sequencing
10
(https://genxpro.net/)
• Degraded products are
ligated with 5’ adapters,
selectively amplified and
sequenced using NGS

11. Workflow
RNA Isolation
Construction of degradome-seq
library
Sequencing by Illumina Hiseq
2500
11

12. Steps
1) Extraction of total RNA from plants
2) Library construction
• mRNA purification (using biotinylated-oligo(dT) probe)
• 5’ adaptor ligation
• RT using oligo dT adaptor
• 3’ adaptor ligation
• PCR amplification
• PAGE - Gel purification
12

13. 13
(Baksa and Szittya, 2017)
Schematic representation of degradome library preparation
Contd…
Steps
MmeI

14. Steps
3) Sequencing and confirmation of miRNA targets
• Illumina base‐calling and quality check (raw data)
• Map to transcriptome
• Summarize data into a "Degradome density file"
• Generate prediction targets (p‐TAREF)
• Compare the degradome density file to the target
predictions, and output significant hits
• Generate "t‐plots" of the targets
14
Contd…

15. 15
(Charles et al., 2008)
Cleavage site 2378
All other sites
target plots (t-plots)
Steps
Contd…
miRNA, corresponding to the
target cleavage site
mRNA
AT1G27370.1 position (nts)
Reads
Score, type, P-value

16. CleaveLand pipeline
Degradome data analysis
16
Sequencing tag
(Addo-Quaye et al., 2009)
Target prediction
mRNA database
Degradome
sequencing
miRNA
High fidelity
miRNA targets

17. Case study 01
OBJECTIVE
Investigate small RNA-mediated nicotine biosynthesisin two tobacco
germplasms,YJ1 and ZY100
17
(Jin et al., 2020)

18. • YJ1 accumulated >2 fold
higher nicotine in the leaves
of different stages than
ZY100
Highlights of the study
18
• Transcriptomes from leaf
and root samples of ZY100
and YJ1 were sequenced
(Jin et al., 2020)
ZY100
YJ1
Nicotine levels at different developmental stages

19. Methodology:
• Identified genes and miRNA by transcriptome analysis and
miRNA sequencing
• Generated reads from samples of ZY100 and YJ1 using
degradome sequencing
• Reads mapped to the tobacco cDNA database
• miRNA targets identified
• Combed in silico and degradome approach
• Generated t-plots
Highlights of the study
19
(Jin et al., 2020)

20. Findings:
• Identified novel miRNA-mRNA pairs as well as miRNAs
targeting to regulation pathway genes
• miRNA-mediated regulatory network of nicotine biosynthesis
was revealed in the tobacco plant
• These newly identified candidates can be used as valuable
tools to dissect nicotine pathway in the future
Highlights of the study
20
(Jin et al., 2020)

21. Case study 02
OBJECTIVE
Analyze seed miRNA expression patterns and their target genes in two
contrasting peanut lines
21
(Ma et al., 2018)

22. Recombinant inbred line (RIL) 8106 has medium-sized pods,
while RIL 8107 has super- large pods
Methodology:
• Constructed two degradome libraries
• Reads mapped to genome
• Identified miRNA cleave targets
• Compared and generated t-plots
Highlights of the study
22
(Ma et al., 2018)

23. • Higher expression levels of the two target genes; NAC100 and
SCL22, were observed in RIL 8107 compared to the levels in
RIL8106
• Identified miRNAs involved in the seed expansion process in
peanut by negatively regulating NAC100 and SCL22
Highlights of the study
23
RIL 8107
NAC100
SCL22
RIL 8106
NAC100
SCL22
(Ma et al., 2018)

24. 24
t-plot of NAC100
(Ma et al., 2018)
RIL8106 RIL8107
nt position nt position
reads
reads

25. 25
(Ma et al., 2018)
t-plot of SCL22
RIL8107
RIL8106
reads
reads
nt position
nt position

26. Findings:
• The read numbers of these miRNAs that cleaved the target
genes in RIL 8107 showed a significant decrease compared to
that in RIL 8106
• The results showed that these two miRNAs maybe involved in
the seed expansion process in peanut by negatively regulating
NAC100 and SCL22
Highlights of the study
26
(Ma et al., 2018)

27. • Further validated expression by RNA-seq and qPCR
• Potential target genes for some conserved and novel miRNAs
were identified by degradome sequencing
• Results provide novel insights into dynamic changes in
miRNAs that occur during peanut seed development
Highlights of the study
27
(Ma et al., 2018)

28. Case study 03
OBJECTIVE
To explore miR164c-guided regulation of seed vigor in rice
28
(Zhou et al., 2022)

29. Materials used:
• Wild-type (WT) indica rice cultivar ‘Kasalath’
• miR164c-silenced line [MIM164c]
• miR164c overexpression line [OE164c]
Treatments:
 No aging treatment (exposed to high temperature & RH)
 Artificial aging treatment
Highlights of the study
29
(Zhou et al., 2022)
It is previously reported that miR164c regulates rice seed vigor

30. Methodology:
• Six degradome libraries were constructed
• Reads compared with the cDNA sequence of the rice
cultivar ‘Nipponbare’ to generate a degradation density
file
• The corresponding target mRNAs:miRNA pair predicted
using TargetFinder
Highlights of the study
30
(Zhou et al., 2022)

31. Findings:
• Number of degradome transcripts of miR164c
Result suggest that miR164c expression was negatively
correlated with seed vigor
Highlights of the study
31
MIM164c
OE164c WT
(Zhou et al., 2022)

32. • An integrative miRNA-mediated gene interaction network
regulating rice seed vigor was uncovered
• Results shed light on the molecular mechanism
underlying seed vigor regulation by miRNAs
Highlights of the study
32
(Zhou et al., 2022)

33. Future prospects
 In vivo mapping of the endoribonucleolytic cleavage sites
 Identification of conserved motifs at the 5ʹ ends of the
uncapped RNA fragments
 Searching for the regions associated with the stacked
ribosomes (or other RNA-binding proteins) on the
transcripts
33

34. Summary
• In plants, miRNAs cause cleavage of their target RNA
• Degradome sequencing provides a comprehensive means
of analyzing patterns of RNA degradation
• Matching cleavage sites to known miRNA sequences links
miRNAs to their targets
• Helps to confirm the miRNA-mediated cleavage of target
transcripts and to identify new target gene
34

35. Conclusion
Degradome sequencing is a powerful technology that can
greatly accelerate the research progress of miRNA-
mediated gene regulation in plants
35

36. THANK YOU