Non-human lnc-DC orthologs encode Wdnm1-like protein

In a recent publication in Science, Wang et al. found a long noncoding RNA (lncRNA) expressed in human dendritic cells (DC), which they designated lnc-DC. Based on lentivirus-mediated RNA interference (RNAi) experiments in human and murine systems, they concluded that lnc-DC is important in differentiation of monocytes into DC. However, Wang et al. did not mention that their so-called “mouse lnc-DC ortholog” gene was already designated “ Wdnm1-like” and is known to encode a small secreted protein. We found that incapacitation of the Wdnm1-like open reading frame (ORF) is very rare among mammals, with all investigated primates except for hominids having an intact ORF. The null-hypothesis by Wang et al. therefore should have been that the human lnc-DC transcript might only represent a non-functional relatively young evolutionary remnant of a protein coding locus. Whether this null-hypothesis can be rejected by the experimental data presented by Wang et al. depends in part on the possible off-target (immunogenic or otherwise) effects of their RNAi procedures, which were not exhaustive in regard to the number of analyzed RNAi sequences and control sequences. If, however, the conclusions by Wang et al. on their human model are correct, and they may be, current knowledge regarding the Wdnm1-like locus suggests an intriguing combination of different functions mediated by transcript and protein in the maturation of several cell types at some point in evolution. We feel that the article by Wang et al. tends to be misleading without the discussion presented here.


Correspondence
In their recent publication in Science, Wang et al. 1 aimed to identify lncRNAs involved in DC differentiation and function. In order to do this they used an established model of human DC differentiation from peripheral blood monocytes (Mo), based on addition of recombinant cytokines. They found that transcription of the human Wdnm1-like pseudogene (Wdnm1-like-ψ), or lnc-DC as they call it, was robustly induced by the Mo-DC differentiation process. Furthermore, they found Wdnm1-like-ψ highly transcribed in other dendritic cells, and confirmed correlation of Wdnm1-like-ψ transcription with DC differentiation in several ways. To investigate a functional role of Wdnm1-like-ψ in their Mo-DC differentiation model, they used a lentivirus-mediated RNA interference (RNAi) system. The RNAi interference with Wdnm1-like-ψ fragments resulted in a pronounced effect on Mo-DC differentiation as measured by expression of genes and molecules involved in the immune system, the ability to take up antigen, and the capacity to stimulate T-helper cells. Wang et al. showed by a number of experiments that the Wdnm1-like-ψ transcript, in particular the 3'-end, has some specificity for binding to the STAT3 transcription factor and can reduce STAT3 dephosphorylation by phosphatase SHP1. And, importantly, they showed that in their human Mo-DC differentiation model the effect of STAT3 inhibition caused similar effects as knockdown of Wdnm1-like-ψ. They therefore postulated that human Wdnm1-like-ψ transcript is an important regulator of DC differentiation by enhancing STAT3 activity through prevention of STAT3 dephosphorylation by SHP1. The results and human model presented by Wang et al. are generally convincing, yet some questions remain, such as to why not for all experiments both "no transfection control" (used in a few experiments) and "control RNAi" (used in all experiments) were included, and why they only used a single RNAi control sequence. RNAi control sequences are relevant because off-target genes might be knocked down (e.g. Jackson et al. 3 ), but also because the lentivirus system using short hairpin RNA (shRNA) can have immunogenic properties in an shRNA-sequencedependent manner (e.g. Kenworthy et al. 4 ). Notably, in some experiments Wang et al. 1 independently knocked down two different fragments of Wdnm1-like-ψ, with similar experimental results, thus reducing the chance that off-target effects of their RNAi systems influenced their conclusions. On the other hand, since the use of two positive RNAi systems suggests that Wang et al. were aware of the potential weaknesses of the system, this raises the question as to why they only used a single sequence for their RNAi control experiments. Regardless, we consider the part of their manuscript on human Wdnm1-like-ψ to be mostly solid and interesting, and the main reason why we are so (overly) critical is that acceptance of the model for human Wdnm1-like-ψ function as proposed by Wang et al. leads to a quite spectacular evolutionary model, as outlined below. Our view, which is supported in the accompanying referee report provided by Dr. Burchard, is that such a spectacular claim requires very robust evidence which in this case probably requires a higher number of RNAi controls.
Whereas the presentation of their human data appears to be mostly correct, we feel that the way Wang et al. 1 present their mouse model is inappropriate. Wang et al. used a mouse model to confirm that knockdown of Wdnm1-like (-ψ) results in impaired DC differentiation. Technically these experiments in mice worked as they expected, indicated both by in vitro and in vivo results, and they also found that knockdown of murine Wdnm1-like could lead to reduction of STAT3 phosphorylation, although they apparently did not check if murine Wdnm1-like transcript can bind STAT3. However, even though Wang et al. refer to Gene symbol 110000G20Rik which mentions "Wdnm1-like", they only present the readers with the term "mouse lnc-DC ortholog". This is highly misleading as it suggests that the transcript also relates to a long noncoding RNA in mice. The authors even state "Taken together, our data suggest that lnc-DC is vital for DC differentiation in both human and mice". However, in mice the gene encodes a functional Wdnm1-like protein, as shown by recombinant analysis 2 , and our extensive analysis of mammalian sequence databases indicates that the Wdnm1-like ORF incapacitation is very rare among mammals. Actually, among the eutherian mammals that we investigated and for which the relevant genomic region information was available, only humans (and Neanderthals and Denisovans) lacked the capacity to encode the otherwise highly conserved Wdnm1-like protein sequence (Figure 1). At the level of the genus Pan (chimpanzee and bonobo) the N-terminus of the predicted mature protein differs from consensus, but even in gorilla and orangutan the encoded Wdnm1-like protein appears fully normal. So possibly the function of the Wdnm1-like protein started to lose importance after separation of Homo/Pan from the other apes, which is quite recent in evolutionary terms. Calculation of synonymous (ds) versus nonsynonymous (dn) nucleotide substitution rates, using software available at http://www.hiv.lanl. gov/content/sequence/SNAP/SNAP.html, indicates conservation of Wdnm1-like protein function after most of the animals shown in Figure 1 had separated in evolution. Namely, in pairwise comparisons, for the depicted set of eutherian mammals except Pan/Homo the average ds/dn ratio is 3.5, and for the set of primates except Pan/Homo this value is 3.0. Thus, although in each individual species experimental evidence would still be required, it is expected that most eutherian mammals possess functional Wdnm1-like protein. Probably because of lack of directed investigations, naturally expressed endogenous full-size Wdnm1-like proteins have not yet been reported. However, our search of the PeptideAtlas database of peptides identified by mass spectrometry identified a rat (Rattus Norwegicus) Wdnm1-like fragment encoded by properly spliced Wdnm1-like transcript (http://www.peptideatlas.org, peptide PAp03984316).
The name Wdnm1-like was first coined by Adachi et al. 5 , who found that Wdnm1-like transcript was differentially expressed in limbal versus central corneal epithelia in rat, and who observed similarity of the encoded protein with Wdnm1. Within the serial analysis of gene expression (SAGE) experiment by Adachi et al. 5 , Wdnm1-like comprised the most abundant SAGE tag present exclusively in the limbal library, and the authors hypothesized that Wdnm1-like might be a marker of limbal stem cells. They could, however, not rule out

Amendments from Version 1
The excellent referee reports were mostly positive, but included a set of extra details and thoughts. For that we now refer to those reports. We now also mention mass spectrometry reports that support the existence of Wdnm1-like protein.

Dipodomys ordii M K L G G F L L L L T L I T L S L E V Q E L Q A A V R P L Q L L G T C M E L C T G D W D C G P G E Q C V S N G C G H V C D T D *
(kangaroo rat) ATGAAGCTGGGAGGCTTCCTTCTTCTGCTGACCCTCATCACCCTCAGCCTGGAAGTGCAGGAGCTTCAGGCTGCCGTGAGACCCCTGCAACTTTTGG GTACCTGCATGGAGCTCTGCACTGGTGACTGGGACTGTGGACCAGGAGAGCAATGTGTCAGCAATGGCTGTGGCCATGTCTGTGACACAGACTAA

Ictidomys tridecemlineatus M K L G G C L L L V A L I F L S L E L Q E L Q A A V R P L Q L L G A C A E L C R G D W D C G P G E H C V S T E C G H D C A S D *
(squirrel) ATGAAGTTGGGAGGCTGCCTTCTCCTTGTGGCACTCATCTTCCTCAGCCTTGAGTTACAGGAGCTTCAGGCTGCAGTGAGACCATTGCAACTTTTGG GGGCCTGTGCTGAGCTCTGCAGAGGTGACTGGGACTGTGGACCAGGGGAGCACTGTGTCAGCACTGAGTGTGGACATGATTGTGCTTCGGACTAA

A V R P L Q L L G T C A E L C K G D W D C G P G E Q C V S S G C S H T C A A S *
(naked mole-rat) ATGAAGTTGGGAAGTCTACTTCTTCTCATGATCCTCATCACCCTCAGCCTAGGGGTGCAGGAGCTACAGGCTGCAGTGAGACCACTGCAACTGTTGG GCACCTGCGCTGAGCTCTGCAAAGGTGACTGGGACTGTGGGCCAGGGGAGCAGTGTGTCAGCAGTGGGTGTAGCCATACCTGTGCTGCAAGTTAA

Cavia porcellus M K L G G L L L L G T L I T L S L V V Q E L Q A A V R P L Q L L G T C I D L C K S D W D C S P E E K C V I H E C S H I C V E R E E H R A S S S P N L V *
(guinea pig) ATGAAGTTGGGAGGCCTCCTTCTTCTCGGGACTCTCATTACTCTCAGCCTAGTGGTACAGGAGCTTCAGGCGGCAGTGAGACCATTGCAGCTGTTGG GCACCTGCATTGACCTCTGCAAAAGTGACTGGGACTGTTCGCCAGAGGAGAAGTGTGTCATCCATGAGTGTAGTCACATCTGTGTTGAACGTGAAGAACATCGGGCCTCTTCTTCCCCAAACCTTGTCTAG Lagomorpha

Oryctolagus cuniculus M N L G G F L L L V T L V T L S V E V Q E L Q A A V R P L Q L F G T C V E L C S G D W D C G V G E S C V S N G C G H V C A T S F S A D K E L S *
(rabbit) ATGAATTTGGGAGGCTTCCTTCTCCTTGTGACCCTCGTCACCCTCAGCGTGGAGGTGCAGGAGCTTCAGGCTGCAGTGAGACCCTTGCAGCTGTTTG GTACCTGTGTTGAGCTATGCAGTGGCGACTGGGACTGTGGAGTGGGAGAGTCCTGTGTGAGCAATGGCTGTGGCCACGTGTGTGCTACAAGCTTTAGTGCAGATAAAGAGTTATCATAA

Ochotona princeps M K L G G F L F L V A L L T L S M E V Q E L E A A V R P L Q L L G P C A E L C R G D W D C E M G E S C V S S G C G H I C V A K *
(pika) ATGAAGTTGGGAGGCTTCCTTTTCCTTGTGGCCCTCCTCACCCTCAGCATGGAGGTACAAGAGCTTGAGGCTGCGGTGAGACCACTGCAACTGCTGG GTCCCTGTGCCGAGCTCTGCAGAGGTGACTGGGACTGTGAGATGGGAGAGTCCTGTGTGAGCAGCGGTTGTGGCCACATCTGTGTGGCCAAATAA Cetartiodactyla

Bos taurus M K L A G F L L L V T L V I L C L V I Q E L Q A A V R P R V F G T C V E L C N G D W D C G P E E R C V S N G C G H V C V *
(cow) ATGAAGTTGGCAGGCTTCCTCCTGCTTGTGACCCTAGTCATCCTCTGCTTAGTCATACAGGAGCTTCAGGCTGCGGTGAGACCATTCAGAGTTTTTG GCACCTGTGTTGAACTCTGCAATGGCGACTGGGACTGTGGGCCAGAAGAGCGCTGTGTCAGCAATGGCTGTGGCCACGTCTGTGTTTAA

Tursiops truncatus M N L G G F L L L V T L V A L S S E V Q E L Q A A M R P L K L L G T C V E L C S G D W D C G P E E H C V R N G C G H E C V S D *
(dolphin) ATGAATTTGGGAGGCTTCCTCCTCCTTGTGACCCTAGTTGCCCTCAGCTCAGAGGTACAGGAGCTTCAGGCTGCCATGAGACCCTTGAAACTTCTGG GCACCTGCGTTGAGCTCTGCAGTGGTGACTGGGACTGTGGTCCAGAGGAGCACTGCGTCAGGAATGGTTGTGGCCACGAATGTGTCTCGGACTAA

Sus scrofa M K L G G F L L L V A L L M L S S E V Q E L Q A A V R P V K F L G S C V D L C H G D W D C D S G E R C V S N G C G H I C V S D *
(pig) ATGAAGTTGGGAGGCTTCCTCCTCCTTGTGGCCCTGCTCATGCTCAGCTCAGAGGTGCAGGAGCTGCAGGCTGCAGTGAGACCAGTGAAATTTCTGG GCTCCTGTGTTGATCTCTGCCATGGGGATTGGGACTGTGATTCAGGAGAACGCTGTGTCAGCAATGGTTGTGGCCACATCTGTGTTTCAGACTAA

Vicugna pacos M K L A G F L L L V T L A I L S S E V Q E L Q A A V R P L K L L G T C V E L C S G D W D C G P E A R C V S N G C G H V C V S D *
(alpaca) ATGAAGTTGGCAGGCTTTCTCCTCCTCGTGACCCTAGCCATTCTCAGCTCAGAGGTACAGGAGCTTCAGGCTGCAGTGAGACCGTTGAAACTTTTGG GCACCTGCGTTGAGCTCTGCAGTGGTGACTGGGACTGTGGTCCAGAGGCACGCTGTGTCAGCAACGGTTGTGGCCACGTCTGTGTTTCAGACTAA Perissodactyla

Equus caballus M K L G G L L L L V T L I T L S L E V Q E L Q A A V R P F H I L G A C V E L C S G D W D C G P E E R C V S N G C G H I C A S V *
(horse) ATGAAGTTGGGAGGCCTCCTCCTCCTTGTGACCCTCATCACCCTCAGCTTGGAGGTACAGGAGCTTCAGGCTGCAGTGAGACCCTTTCATATTTTGG GCGCCTGTGTTGAGCTCTGCAGCGGTGACTGGGACTGTGGCCCAGAGGAGCGCTGTGTCAGCAACGGCTGTGGCCACATCTGTGCTTCAGTCTAA

Ceratotherium simum M K L G S L L L L V T L I I L S L E V Q E F Q A A V R P F R L L G T C V E L C N G D W D C D P G E H C I S N G C G H V C A S G *
(rhinoceros) ATGAAGTTAGGAAGCCTCCTCCTCCTTGTGACCCTCATCATCCTCAGCTTAGAGGTACAAGAGTTTCAGGCTGCAGTGAGACCATTTCGTCTTTTGG GCACCTGTGTCGAGCTCTGCAATGGTGACTGGGACTGTGATCCAGGAGAGCACTGCATCAGCAACGGGTGTGGCCATGTCTGTGCTTCAGGCTAA Chiroptera

L S S E V Q E L Q A A V R P L R L L G T C V E L C T N D E D C D L G E H C V S N G C G H I C A P *
(dog) ATGAAGTTGGGGGGATTCCTCCTTCTGGTGATGCTTCTCACCCTCAGCTCAGAGGTACAGGAGCTCCAGGCTGCGGTGAGACCATTGAGACTTCTAG GAACCTGCGTTGAGCTCTGCACAAATGACGAGGATTGTGATCTAGGAGAGCACTGTGTCAGCAATGGGTGTGGCCACATCTGTGCTCCAGCCTAA

Ailuropoda melanoleuca M K L G G F F L L V M L I T L S S E V Q Q L Q A A V R P L R V L G T C I E L C S D D W D C D L G E H C V S N G C G H I C A A A *
(panda) ATGAAGTTGGGGGGCTTCTTCCTCCTGGTGATGCTCATAACCCTCAGCTCGGAGGTCCAGCAGCTCCAGGCTGCGGTGAGACCACTCCGAGTTCTGG GCACCTGCATTGAGCTCTGCAGTGATGACTGGGATTGTGATCTGGGGGAGCACTGCGTCAGCAATGGGTGTGGCCACATCTGTGCTGCAGCCTAA

Orycteropus afer M K L G G F L I L V T L L A L S L E V Q D L Q A A V R P V Q I L G T C V E H C S S D W D C E A G E R C V S N G C G H I C K S N *
(aardvark) ATGAAGTTGGGAGGCTTCCTCATCCTTGTGACCCTCCTTGCCCTCAGCCTAGAGGTGCAGGACCTGCAGGCGGCTGTGAGACCCGTGCAAATTCTGG GCACCTGCGTAGAACACTGCAGCAGTGACTGGGACTGTGAGGCAGGGGAGCGATGTGTCAGCAATGGATGTGGCCACATCTGCAAATCCAACTAA Xenarthra After evolutionary separation from gorilla, in an ancestor common to the genera Pan (including chimpanzee and bonobo) and Homo (including human, Neanderthal and Denisovan), the nucleotide region coding the N-terminus of the mature Wdnm1-like protein was modified by deletions (yellow shading). Nevertheless, in the genus Pan the Wdnm1-like open reading frame (ORF) remained intact. Only in Homo the Wdnm1-like coding sequence was interrupted by a frameshift through a single nucleotide deletion (red shading) within the leader peptide coding region (the resulting change in amino acids is shaded grey). For the human Wdnm1-like locus several transcripts (splicoforms) were found (Ensembl reports ENST00000590346, ENST00000588180, ENST00000587298, ENST00000590012, ENST00000589987, ENST00000592556, ENST00000566140, and ENST00000589777); however, we agree with Wang et al. 1 that software investigation of the known transcripts suggests that the human Wdnm1-like locus does not code a functional protein (analyses not shown).

Dasypus novemcinctus M K L G G F L L L V A L L T L S L E A Q Q L Q A A A T P L R I V G A C V E F C S S D L D C D T G E R C V S N G C G H I C A A E *
The marsupial Monodelphis domestica (opossum) was the only non-eutherian mammal for which we could identify Wdnm1-like, situated upstream of the gene HEAT Repeat Containing 6 (HEATR6) like its ortholog in eutherian mammals. To avoid gaps in the bulk of the figure, the N-terminus of the opossum sequence is not perfectly aligned with Wdnm1-like of eutherian mammals. Except for rabbit (see Methods section), the figure shows the ORFs of sequences corresponding to the murine Wdnm1-like protein coding transcript of NCBI accession NM_183249, while other (possible) splicoforms are neglected. The intron site is indicated by a downward triangle. Intron sequences are not shown, but the below listed genomic sequence reports agree with GT-AG borders. For most of the species, the depicted sequences were supported by transcript reports, as exemplified per species in the Methods section. In the figure, dashes indicate gaps that were introduced for optimal sequence alignment. The alignments were performed by hand. Amino acid sequences are indicated above the second nucleotides of codons. Basic residues are indicated in red, acidic residues in blue, and green residues are more hydrophilic than the orange ones (following reference 9 ). Cysteines are in violet. Asterisks correspond with stop codons. Predicted leader sequences are underlined.
The mouse Wdnm1-like sequence was designated "mouse lnc-DC ortholog" by Wang et al. 1 , and they targeted the regions shaded blue and green for transcript knockdown by "RNAi-1" and "RNAi-2", respectively, using a lentivirus-mediated RNA interference system.
the possibility that Wdnm1-like was expressed by other cell types present in limbal epithelia, such as for example dendritic cells. A later study on rodent Wdnm1-like was performed in mice by Wu and Smas 2 . Wu and Smas got interested in Wdnm1-like after they found it highly upregulated upon differentiation of preadipocytes into adipocytes. They found Wdnm1-like to be selectively expressed in liver and adipose tissue, and enriched in white adipose depots versus brown. Recombinant expression of tagged murine Wdnm1-like in HT1080 human fibrosarcoma cells revealed a small secreted protein 2 . Because Wdnm1-like is a distant member of the whey acidic protein/four-disulfide core (WAP/4-DSC) family, of which several members have roles as proteinase inhibitors, Wu and Smas speculated that Wdnm1-like might have a similar function. An important class of extracellular proteases involved in adipocyte differentiation are the matrix metalloproteinases (MMPs), which can degrade extracellular matrix (ECM) components. Therefore, Wu and Smas investigated whether MMPs expressed by HT1080 were affected by the recombinant Wdnm1-like expression, and they found an increased amount of the active form of MMP-2 2 . Thus, rather than having an inhibitory effect, Wdnm1-like appears to enhance activation of a protease. Wu and Smas conclude with "Future studies are required to address the mechanism(s) underlying the function and regulation of adipocyte-secreted Wdnm1-like" 2 , and according to literature this situation has not changed since then.
Looking at the combined publications, a very complicated picture emerges. In most mammals the Wdnm1-like locus encodes a protein, with humans as an exception which is possibly unique. In rat, Wdnm1-like is differentially expressed in limbal versus central corneal epithelia 4 . In mouse, Wdnm1-like is expressed upon adipogenesis, and Wdnm1-like protein enhances the production of active MMP-2 2 . In human and mouse, the Wdnm1-like(-ψ) transcript appears functionally associated with dendritic cell differentiation, and at least in humans this may be mediated by binding of the transcript to STAT3 1 . This leaves questions for future research such as, for example, whether human Wdnm1-like-ψ transcript is also associated with adipogenesis, and whether murine Wdnm1-like transcript exerts its function on DC differentiation by binding to STAT3 or by encoding Wdnm1-like protein. Supporting that the Wdnm1-like proteins and transcripts in some extinct or extant animals may have (had) synergetic functions, is the fact that differentiation of both adipocytes and limbal epithelial cells can involve STAT3 6,7 . So, despite our points of criticism, we think that the results and human model by Wang et al. may be valid and part of a more complex evolutionary scenario that involves distinct functions at the transcript and protein level, and a number of different tissues and cell types. In general, we think that studies on long noncoding RNAs typically require discussion of the evolutionary context 8 , especially when dealing with wide species borders such as between human and mouse.
Additional note 1 A nice speculation allowed by the combined referenced articles is that Wdnm1-like protein might promote Mo-DC differentiation in humans. After all, murine Wdnm1-like protein was found to enhance MMP-2 activity of human HT1080 cells 2 , concluding that humans did not lose their sensitivity to Wdnm1-like protein.

Additional note 2
We did not feel comfortable with the amount of space and visibility the editors of the journal Science were able to offer us via their commenting mechanism for the discussion presented here. Therefore, we declined their offer, and instead deemed publication in F1000Research a more appropriate vehicle. Through F1000Research we asked specialists and the corresponding authors of several of the referenced articles, including the article by Wang et al., to provide referee reports (which may also include broad views) on our discussion. We are pleased with the excellent referee reports received from Dr. Smas and Dr. Ren, and Dr. Burchard, and recommend them to the readers of our article. Importantly, Drs. Smas and Ren confirmed that we correctly summarized reports on rodent Wdnm1-like and listed additional evidence to that matter, while Dr. Burchard substantiated and detailed our notion that the RNAi experiments by Wang et al. were inconsistent and probably incomplete. We would welcome comments from the group of Wang et al. and also encourage other researchers to leave comments.
Additional note 3 Wdnm1-like protein appears to be very interesting. Not only may it be involved in differentiation of several cell types, it also is intriguing because it appears highly conserved throughout eutherian mammals and (rather) uniquely lost in hominids. It may help determine what makes us human.

Methods
The partial Wdnm1-like sequence information available for extinct hominids, namely, for Neanderthal and Denisovan, was retrieved using the UCSC genome browser (http://genome.ucsc.edu). All other sequences shown in the figure were retrieved from Ensembl (www.ensembl.org/) or NCBI (http://www.ncbi.nlm.nih.gov/) databases. For a representative list of model species, we investigated database sequences of all mammals for which genomic sequences are available in the Ensembl database, and also of Pan paniscus (bonobo). For some of those animals sequence information for the Wdnm1-like ORF or for its expected genomic site was incomplete, and in such case the sequence is not included in the alignment figure.

Homo sapiens (human)
The

Tupaia chinensis (Chinese tree shrew)
The depicted Chinese tree shrew Wdnm1-like sequence maps within Ensembl database TREESHREW, scaffold_15853 positions 2941-to-6216, reverse orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl|SRA|SRR518934.53798716.1 of experiment set SRX157966.

Microtus ochrogaster (prairie vole)
The depicted prairie vole Wdnm1-like sequence maps within Ensembl database MicOch1.0, Chr.7 positions 15310620-to-15312860, reverse orientation. According to the Ensembl database the prairie vole also has an intronless copy of Wdnm1-like gene on Chr.X (not shown). Transcription is supported by NCBI SRA database sequence reports, such as for example gnl|SRA|SRR058428.108679.2 of experiment set SRX018513.

Oryctolagus cuniculus (rabbit)
The depicted rabbit Wdnm1-like sequence maps within Ensembl database OryCun2.0, Chr.19 positions 25079843-to-25084775, forward orientation. The underlined part in Italic font at the 3'end belongs to a third exon. Transcription is supported by for example NCBI accession gb|GBCH01008538.1|.
Transcription is supported by NCBI SRA database sequence reports, such as for example gnl|SRA|SRR1013468.27145136.1 of experiment set SRR1013468.

Felis catus (cat)
The depicted cat Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb|AANG02057756.1|, positions 9507-to-13123, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl|SRA|SRR835496.27404932.1 of experiment set SRX272142.

Monodelphis domestica (opossum)
The Author contributions JMD did most of the research and wrote the manuscript. KTB analyzed sequence databases of extinct hominids and carefully checked the manuscript.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in the funding of this work.

2.
3.  A few items of follow-up may be of interest to clarify and extend the evolutionary observations in this correspondence.

Open Peer Review
Is the frameshift in the erstwhile signal peptide coding sequence in the human reference genome reproduced in all 1000-genomes data, or is WFDC21P a polymorphic human pseudogene?
Is guidance by the human reference sequence in assembly of and genomes Neanderthal Denisova a potential factor in their reproduction of the reported human frameshift?
Is there human genetic variation at this locus tied to variation in trait expression, or alternatively is there evidence that variation in this small gene is sufficiently suppressed to leave no functional variation for genetic association studies to mine? Either could be consistent with a significant role in immune function as proposed by Wang et al. What is found at the position syntenic to Wfdc21 in marsupials other than the one noted as sharing this gene, and in lower model organisms? This may help clarify the nature of the apparent mammalian innovation at this locus.
Do regulatory elements for WFDC21P differ between hominids and other species? If Wfdc21 is a 5.

4.
Do regulatory elements for WFDC21P differ between hominids and other species? If Wfdc21 is a gene with active RNA and protein products, their function will have evolved in the context of cell-type specific expression. Fantom5 CAGE tag data suggests a difference in regulation of human WFDC21P vs. mouse Wfdc21. Although parallel samples are not available for all tissues and cell types, mouse data show strongest expression in myeloid suppressor cells with significant expression also in liver and skin, while human data show 1000x lower maximum expression with best expression in migratory Langerhans cells. The correspondents comment on the inconsistent use of controls. Indeed, the sole controlled experiment suggesting RNA-protein association appears to be the pulldown of STAT3 with biotinylated WFDC21P RNA, with specific absence of the STAT3 band in the antisense control. While RIP with STAT3 experimental and STAT1 control antibodies was conducted, no sequencing is reported so the specificity of interaction with STAT3 is not known. Further, figures on RNA-FISH visualization of association of STAT3p with WFDC21P-RNA do not show controls.

Wang
rely on inhibitors to demonstrate WFDC21P-RNA function. As the correspondents et al. note, one shRNA sequence is primarily employed and it is not consistently paired with varied on-or off-target controls. Literature on functional siRNA screens suggests that a half dozen RNAi sequences with independent seeds are required for dissociation of off-and on-target activities. Further, Wang have performed expression profiling on shRNA-treated cells. The profiles can et al. be examined for seed-based off-target activity and for inflammatory response to the lentiviral vector according to published methods. It will be important to establish whether the dendritic cell proteins whose differential expression is highlighted by Wang show shRNA-matching seed et al. sequences in their 3'UTRs or respond to lentiviral infection.

Wang
also use published STAT3 inhibitors to elucidate the role of WFDC21P-RNA. It would et al. be intriguing to speculate that an RNA-protein interaction site helps to define the STAT3 binding site of published inhibitor S3I-201 and its effects on STAT3 activity. However, the supplementary material provided by Wang show much more profound effects on cytokine production by et al. small molecules than shown for WFDC21P shRNA in the main paper, although effects on T-cell activation remain similar.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. We thank you for your extensive and valuable comments. Like the comments by Drs. Smas and Ren, we embrace them as generally positive. Your comments add accuracy to our story, especially regarding the technical part of the RNA investigation by Wang and co-workers.
You wonder, as do Drs. Smas and Ren, and as do we, whether in some individuals or under some conditions, humans may express Wdnm1-like protein. After all, the sequence for the mature protein appears intact, and requirements for a leader peptide are not very unique. However, our rather extensive database investigations could not retrieve a human sequence expected to encode a functional Wdnm1-like protein. Identical frameshifts in Neanderthal and Denisovan Wdnm1-like sequence reports, for the reliability of which we have to depend on the respective authors, argue against the likeliness of functional/nonfunctional Wdnm1-like polymorphism in modern humans. An expressed sequence tag (EST), reported as GenBank accession CD692402, suggests that an individual from southern China may have a protein coding sequence; however, Wdnm1-like besides repair of the frame-shift in the leader coding region, this sequence has an additional unique modification, and the sequence report may contain technical errors. In short, we could not obtain evidence for intact Wdnm1-like coding sequences in humans, but cannot exclude the possibility that such sequences exist. We would welcome if anyone could provide such evidence or indications.
We took a brief look at Wdnm1-like evolution beyond eutherian mammals. However, except for the mentioned case in opossum, at this evolutionary distance it becomes difficult to distinguish Wdnm1-like orthologues from other family members, and it would become more a discussion on the evolution of Wdnm1-like plus related molecules than of Wdnm1-like alone. Because the primary goal of our study is the discussion of the Wang article, which is confined to eutherian et al. mammals, we feel a discussion of deeper Wdnm1-like evolution would make our study too complicated.
Although we did not make systematic comparisons among various genes, we feel that overall the 3'-end region of human transcript which is believed to interact with STAT3, is not Wdnm1-like-y especially well conserved among mammals. However, without knowing the precise sequence motif or RNA secondary structure important for that binding, we probably shouldn't speculate on presence or absence of evolutionary constraints on that region.
You raise four additional points regarding the technical merits of the work presented by Wang and co-workers. Importantly, you agree with us that the Wang study was inconsistent and et al. probably incomplete in the use of controls. Some points you raise are valid speculations and questions, whereas others can be considered as criticism of the Wang article. In our opinion et al. that criticism is mostly right. However, we prefer not to change the open style of our technical comments, and hope that the readers will find the specific issues that you raise when reading your report.
Thank you again for your hard and valuable work.

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Given this, it was decided that this laboratory (Smas) would not go further on the project in respect to the study of the Wdnm1-like protein. As such, the human gene or transcript has not been addressed in studies from this laboratory (Smas), and we not aware of any data reporting on expression of this transcript in human adipose tissue. However, while not the subject of such studies, murine Wdnm1-like has been mentioned within several publications on adipocytes and adipose tissue . These relate to the finding that the Wdnm1-like transcript is highly enriched in expression in white brown murine vs. adipocytes/adipose tissue . Of interest, knockout mice for Wfdc21 (Wfdc21 ) are now available by embryo resuscitation through the KOMP project (Project ID: ) and would serve as CSD49368 a highly useful system in which to address the role of Wdnm1-like in DC cell maturation in mice.
In the prior publication on Wdnm1-like from this laboratory (Smas), our studies utilized a murine Wdnm1-like expression construct with a C-terminal epitope tag, and cell transfection studies. These showed that a protein of predicted size for Wdnm1-like was ectopically expressed and secreted into culture media. Dijkstra and Ballingall do accurately describe our studies and clearly state that our work on Wdnm1-like utilized recombinant ectopic expression of the predicted murine Wdnm1-like open reading frame. However, one would have liked to have seen Dijkstra and Ballingall make it even more clear, earlier on in their text, that the endogenous Wdnm1-like protein has not yet been demonstrated in any system/species. A quick Google search fails to find an available antibody to the Wdnm1-like protein, so the endogenous protein remains to be investigated.
The work by Dijkstra and Ballingall makes several very important points: It provides well-needed clarification and extensive documentation that apparently only for hominids does the locus for encode a long non-coding RNA, while in all other species Wdnm1-like specifically examined (which was quite extensive) this locus contains an open reading frame for the Wdnm1-like protein.
It raises concerns as to the ultimate responsibility of Wang and co-authors to report the full range of information on such distinctions within their report, if they indeed were aware of such. Science Only Wang and co-authors can inform us of their extent of knowledge of the protein coding nature of murine human at the time of their publication. Thus it is not possible at vs.
Wdnm1-like Science this point to know whether such information was selectively omitted. But as Dijkstra and Ballingall point out, Wang and co-authors refer to the GenBank entry for murine Wdnm1-like , also known as 100001G20Rik and now formally named Wfdc21. This GenBank entry contains citations for publications on murine Wdnm1-like . It seems very odd if Wang and co-authors were not aware of the distinctions between the human and murine forms of , particularly in today's age Wdnm1-like of well-curated databases. In fact, the NCBI Unigene entry for this gene reveals the human version is annotated as pseudogene (LOC645638, Hs.463652). Perhaps this was one more Wdnm1-like instance of a research group "rediscovering and renaming" a gene that was previously published on. This is all too common of late; doing so essentially tosses aside the already peer-reviewed and published work of others . However, until further clarification is forthcoming by Wang and co-authors, one would hope they would be provided with the benefit of the doubt on the facts and intentions in regard to this matter.
A third valid concern raised by Dijkstra and Ballingall, is whether the knowledge that the murine gene is predicted to be protein encoding, while the human gene encodes a long non-coding RNA ( ), impacts the quality or interpretation of the data in the manuscript by Wang and co-authors. lnc-DC It appears that for the vast majority of the studies in their publication, Wang and co-authors Science utilized human cell culture systems, and limited studies were conducted in murine systems. Thus we are in agreement with Dijkstra and Ballingall that the essential conclusions of the report Science are not dependent on the murine studies, or the fact that encodes a long non-coding Wdnm1-like 4-6 4-6 tm1a(KOMP)Wtsi 1,7 1,7