MicroRNAs and their isomiRs function cooperatively to target common biological pathways.
BACKGROUND
Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules.


RESULTS
To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs.


CONCLUSIONS
Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.
Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia. n.cloonan@imb.uq.edu.au
Nicole
N
Cloonan
Shivangi
S
Wani
Qinying
Q
Xu
Jian
J
Gu
Kristi
K
Lea
Sheila
S
Heater
Catalin
C
Barbacioru
Anita L
AL
Steptoe
Hilary C
HC
Martin
Ehsan
E
Nourbakhsh
Keerthana
K
Krishnan
Brooke
B
Gardiner
Xiaohui
X
Wang
Katia
K
Nones
Jason A
JA
Steen
Nicholas A
NA
Matigian
David L
DL
Wood
Karin S
KS
Kassahn
Nic
N
Waddell
Jill
J
Shepherd
Clarence
C
Lee
Jeff
J
Ichikawa
Kevin
K
McKernan
Kelli
K
Bramlett
Scott
S
Kuersten
Sean M
SM
Grimmond
D060565:Argonaute Proteins / Q000235:genetics*; D001483:Base Sequence; D019921:Biotinylation; D053487:DEAD-box RNA Helicases / Q000235:genetics; D020869:Gene Expression Profiling; D053263:Gene Regulatory Networks / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics*; D043244:Ribonuclease III / Q000235:genetics; D016415:Sequence Alignment; D059467:Transcriptome; D014162:Transfection
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases