Individual long non-coding RNAs have no overt functions in zebrafish embryogenesis, viability and fertility

  1. Mehdi Goudarzi  Is a corresponding author
  2. Kathryn Berg
  3. Lindsey M Pieper
  4. Alexander F Schier  Is a corresponding author
  1. Harvard University, United States
  2. University of Washington, United States
  3. University of Basel, Switzerland
25 figures, 1 table and 3 additional files

Figures

Figure 1 with 4 supplements
Genomic location of selected lncRNAs.

The chromosomal positions of selected lncRNAs are depicted. lncRNAs discussed in the text are underlined. The corresponding genomic coordinates for all lncRNAs are provided in the supplementary file …

https://doi.org/10.7554/eLife.40815.003
Figure 1—figure supplement 1
Size, relative distance and orientation of selected lncRNAs and their neighboring genes

(A) lncRNA names and sizes are shown in the middle section (blue columns). The distance, size and transcriptional orientation of the neighboring genes, in a 200 kb window centered on lncRNA’s TSS …

https://doi.org/10.7554/eLife.40815.004
Figure 1—figure supplement 2
Expression levels of selected lncRNAs and their neighboring protein-coding genes.

LncRNAs are color coded as blue (Intergenic), brown (Overlapping) and green (Divergent/Promoter associated) (see Figure 1—figure supplement 1B). For each lncRNA and its upstream (top) and downstream …

https://doi.org/10.7554/eLife.40815.005
Figure 1—figure supplement 3
Cas9-mediated deletion approach for generating lncRNA knockouts 6 gRNAs (three at either side of the TSS) were used to remove TSS.

Nine guide RNAs (the first six plus three additional gRNAs around the Transcriptional Termination Site, TTS) were used to generate the gene deletions. Relative positions of genotyping primers are …

https://doi.org/10.7554/eLife.40815.006
Figure 1—figure supplement 4
Summary of qRT-PCR analysis for lncRNA and their neighboring genes.

Visual representation of the expression level changes for each lncRNA and its neighboring genes in homozygous deletion mutants. Three biological replicates for homozygous mutant and wild-type …

https://doi.org/10.7554/eLife.40815.007
Normal embryogenesis of cyrano mutants.

(A) The positions of TSS-deletion allele and gene deletion allele are marked by dashed red lines. Green box represents the conserved element in cyrano which is complementary to miR-7. Solid red …

https://doi.org/10.7554/eLife.40815.008
Normal embryogenesis of gas5 mutants.

(A) Position of the TSS-deletion allele in gas5 is marked by dashed red line. Arrows flanking black dotted lines mark the primer binding sites for 5’-qPCR and 3’-qPCR products. (B) Representative in …

https://doi.org/10.7554/eLife.40815.009
Normal embryogenesis of lnc-setd1ba mutants.

(A) The relative position of lnc-setd1ba and the protein-coding gene setd1ba. The gene deletion region is marked by dashed red line. Arrows flanking black dotted line mark the primer-binding sites …

https://doi.org/10.7554/eLife.40815.010
Figure 5 with 1 supplement
No non-coding function for squint 3’UTR.

(A) The position of untranslated regions (brown), coding region (green), putative Dorsal Localization Element- DLE (blue) and the gene deletion (red dashed line) in the squint genomic locus. Arrows …

https://doi.org/10.7554/eLife.40815.011
Figure 5—figure supplement 1
Dorsalization induced by Overexpression of squint mRNA but not its non-protein coding version.

(A) Schematic representation of injected mRNAs. Cap-analog is indicated by in blue circles at the beginning of each mRNA. squint non-protein coding mRNA was generated by adding 8 Adenine-nucleotides …

https://doi.org/10.7554/eLife.40815.012
Requirement for lnc-phox2bb genomic elements but not RNA.

(A) The red dashed lines depict the respective positions of the lnc-phox2bb TSS and gene deletion. Arrows flanking black dotted line mark the primer binding sites for qRT-PCR product. (B) Homozygous …

https://doi.org/10.7554/eLife.40815.013
Author Response image 4
Clear enrichment in the margin and prechordal plate cells.
https://doi.org/10.7554/eLife.40815.021
Author Response image 5
Some partial enrichment in the Neural Plate Border cells and Somites.
https://doi.org/10.7554/eLife.40815.022

Tables

Table 1
Summary of lncRNA features and mutant phenotypes lncRNA names are shown in the first column.

lncRNAs were named using the last four digits of their corresponding ENSEMBL Transcript ID or their chromosome number if no transcript ID was available (e.g. lnc-1200 is located on chromosome 12). …

https://doi.org/10.7554/eLife.40815.002
lncRNA mutant,
deletion type
Ribosome
Profiling,
class
lncRNA
transcript ID
Deletion
size
Percent
reduction
Embryonic
phenotype
Viability and fertilityNeighboring
genes
Selection
criteria
Up 100 KbDown 100 Kb
cyranoa171,
TSS-del.
TrailerlikeENSDART00000139872326 bp98%NoYestmem39boip5Syntenic and sequence
conservation,
Reported phenotype
cyranoa172,
gene del.
TrailerlikeENSDART000001398724374 bp94%NoYestmem39boip5Syntenic and sequence conservation,
Reported phenotype
gas5a173,
TSS-del.
LeaderlikeENSDART00000156268296 bp100%NoYesosbpl9tor3aSyntenic conservation,
well studied lncRNA,
host of several snoRNA
lnc-setd1baa174,
gene del.
LeaderlikeENSDART000001415003137 bp100%NoYessetd1barhoFSyntenic and sequence conservation,
Proximity to developmental regulatory genes
squinta175,
gene del.
CodingENSDART0
0000079692
1032 bp95%NoYeshtr1abeif4ebp1Evolutionary conservation,
Reported phenotype,
putative cncRNA
lnc-phox2bba176,
TSS-del.
LeaderlikeENSDART00000158002652 bp99%NoYessmntl1phox2bbSyntenic conservation
lnc-phox2bba177,
gene del.
LeaderlikeENSDART000001580029361 bp87%YesNosmntl1phox2bbSyntenic conservation
lnc-3852a178,
TSS-del.
LeaderlikeENSDART00000153852447 bp100%NoYeslima1ahoxc1aMaternal expression,
Proximity to developmental
regulatory genes
lnc-1562a179,
TSS-del.
LeaderlikeENSDART00000131562409 bp90%NoYes*fgf10aMaternal expression,
Proximity to developmental
regulatory genes
lnc-3982a180,
TSS-del.
LeaderlikeENSDART00000153982352 bp97%NoYes*bmp2bMaternal expression,
Proximity to developmental
regulatory genes
lnc-6269a181,
TSS-del.
LeaderlikeENSDART00000156269535 bp99%NoYestbx1*Maternal expression,
Proximity to developmental
regulatory genes
lnc-2154a182,
TSS-del.
TrailerlikeENSDART00000132154546 bp100%NoYesrpznr2f5Maternal expression,
Proximity to developmental
regulatory genes
lnc-1200a183,
TSS-del.
LeaderlikeChr12:1708389-1925779:1590 bp95%NoYes*zip11Maternal expression,
Longest
selected lncRNA
lnc-1200a184,
gene del.
LeaderlikeChr12:1708389-1925779:1203.8 kb84%NoYes*zip11Maternal expression,
Longest selected lncRNA
lnc-2646a185,
TSS-del.
LeaderlikeENSDART00
000152646
240 bp97%NoYes*dkk1bProximity to developmental
regulatory genes
lnc-4468a186,
TSS-del.
LeaderlikeENSDART00000154468306 bp100%NoYesfam169ablhx5Proximity to developmental
regulatory genes,
Low expression level
lnc-0600a187,
TSS-del.
TrailerlikeChr6:59414652-59443141:1244 bp95%NoYes*gli1Proximity to developmental regulatory
genes,
Low expression level
lnc-0900a188,
TSS-del.
LeaderlikeChr9:6684669-6691350:1377 bp83%NoYespou3f3a*Syntenic conservation,
Low expression
level
lnc-8507a189,
mTSS-del.
LeaderlikeENSDART00000158507323 bp81%NoYesnpvfhoxa1aProximity to Hox genes,
Maternal and Zygotic
promoters
lnc-8507a190,
mzTSS-del.
LeaderlikeENSDART000001585079773 bp95%NoYesnpvfhoxa1aProximity to Hox genes,
Maternal and Zygotic
promoters
lnc-7620a191,
TSS-del.
TrailerlikeENSDART00000137620668 bp99%NoYesgal3st1bsrsf9Syntenic and sequence conservation,
Implicated in adult fish
and mouse behavior.
Bitetti, A., et al. (2018)
lnc-1300a192,
TSS-del.
LeaderlikeChr13:4535992-4538275:1367 bp92%NoYesc1dpla2g12bSyntenic and sequence conservation,
High expression
level
lnc-7118a193,
TSS-del.
TrailerlikeENSDART00000157118438 bp82%NoYesmrps9pou3f3bSyntenic conservation
lnc-5888a194,
TSS-del.
LeaderlikeENSDART00000155888606 bp96%NoYesglrx5zgc:100997Syntenic conservation,
scaRNA13 host gene,
shortest selected lncRNA
lnc-6913a195,
TSS-del.
LeaderlikeENSDART00000156913333 bp72%NoYesusp20ptgesProximity
to developmental regulatory
genes
lnc-6913a196,
gene del.
LeaderlikeENSDART000001569135568 bp93%NoYesusp20ptgesProximity
to developmental regulatory
genes
lnc-1666a197,
TSS-del.
LeaderlikeENSDART00000141666544 bp96%NoYesptf1a*Proximity to developmental
regulatory genes, Restricted
late expression
lnc-6490a198,
TSS-del.
LeaderlikeENSDART00000146490607 bp99%NoYesnr2f2*Syntenic conservation,
Restricted late expression
lnc-6490a199,
gene del.
LeaderlikeENSDART000001464908378 bp100%NoYesnr2f2*Syntenic conservation,
Restricted
late expression
lnc-0464a200,
TSS-del.
TrailerlikeENSDART00000140464597 bp96%NoYesnr2f1a*Restricted late expression
pattern
lnc-4149a201,
TSS-del.
LeaderlikeENSDART00000154149491 bp98%NoYesbhlhe22*Proximity to developmental
regulatory genes
lnc-4149a202,
gene del.
LeaderlikeENSDART0000015414935.11 kb100%NoYesbhlhe22*Proximity to developmental
regulatory genes

Additional files

Supplementary file 1

This compressed folder contains three Excel files for the sequences of gRNAs, genotyping and qRT-PCR primers (for lncRNAs and their neighboring genes) and also the annotated sequence files (.ape) for each lncRNA and their deleted segments.

https://doi.org/10.7554/eLife.40815.014
Supplementary file 2

This genome-browser-compatible file is in the bed formant, containing the coordinates for all the lncRNAs investigated in this manuscript based on the GRCz11 (GCA_000002035.4).

https://doi.org/10.7554/eLife.40815.015
Transparent reporting form
https://doi.org/10.7554/eLife.40815.016

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