You'd Prefer An Argonaute

Secreted Monocytic miR-150 Enhances Targeted Endothelial Cell Migration

Yujing Zhang, Danqing Liu, Xi Chen, Jing Li, Limin Li, Zhen Bian, Fei Sun, Jiuwei Lu, Yuan Yin, Xing Cai, Qi Sun, Kehui Wang, Yi Ba, Qiang Wang, Dongjin Wang, Junwei Yang, Pingsheng Liu, Tao Xu, Qiao Yan, Junfeng Zhang, Ke Zen, and Chen-Yu Zhang

Molecular Cell 39, 133–144, 9 July 2010.
DOI: 10.1016/j.molcel.2010.06.010

This week’s summary and gloves-off analysis by Anonymous:

This group had previously examined microRNA (miRNA) profiles in the serum samples of patients with certain cancers and diabetes, and found them to be able to serve as biomarkers for these diseases (Chen et al, 2008). In that study, they also found that serum miRNAs were resistant to RNase A digest and this study follows up on that. Exosomes/microvesicles (MVs) are small vesicles shed from many cell types of endocytic origin. These are delimited by a lipid bilayer and have been found to contain proteins, mRNAs and miRNAs. MVs can deliver their contents to recipient cells and while it has been shown previously that delivered proteins can alter cellular functions in recipient cells (Skog et al, 2008; Valadi et al, 2007), there has been no direct evidence of miRNAs being delivered to alter target gene expression in recipient cells. This study thus set out to fill that gap.

Briefly, the group first shows that MVs generated by THP-1 cells (a human macrophage/monocytic cell line) contained miRNAs that were resistant to RNase A digest by virtue of the protection afforded by the MV membrane. Next, the authors attempted to show that upon treatment by various stimuli, cellular miRNAs are selectively packaged into MVs such that the miRNA profile in MVs differs from that in the origin cells. However, the evidence was not convincing. The entire study uses quantitative real-time PCR (qRT-PCR) to measure miRNA expression levels. Aside from concerns that qRT-PCR measurements of miRNAs can be wildly noisy, this study is also handicapped by the fact that a reliable internal control that can be found in both cells and MVs is hard to find (it is unclear which control was used in this study, if any). Although the authors attempted to get around this issue by measuring absolute levels of miRNAs normalized to the total protein content in MVs, the miRNA levels in the “no-treatment control” for three different sets of stimuli are not very comparable (even though they should be if absolute levels were measured), underscoring the noise inherent in the miRNA qRT-PCR and/or normalization method. As such, it cannot be said conclusively that miRNAs are selectively packaged into MVs upon different stimulation. It would have been better if the authors had used deep sequencing to quantify miRNA expression instead.

It is, however, fair to say that MVs from THP-1 cells contain high levels of miR-150, which can be delivered to recipient HMEC-1 cells (an endothelial cell line). Upon incubation with THP-1 MVs, miR-150 levels (originally low in HMEC-1 cells) were increased in the recipient cells. The authors also checked that this was not because interactions with the MVs caused the HMEC-1 cells themselves to upregulate expression of miR-150 by checking the levels of pre-miR-150 (which were unaltered) in the HMEC-1 cells. The delivered miR-150 was shown to repress the protein levels of c-Myb, a known miR-150 target, in HMEC-1 cells, and this downregulation enhanced the migration capability of the HMEC-1 cells. Numerous controls were done here to demonstrate that this effect could only be seen when the donor MVs came from cells with high levels of miR-150, which is perhaps the redeeming factor in this paper. Although the authors showed that miR-150 repressed c-Myb protein expression via the 3′ untranslated region (3′UTR), they did not mutate the miR-150 target sites in the 3′UTR to show direct targeting definitively. The paper ends by showing that MVs that were intravenously injected into mouse tail veins can be taken up by the endothelium of mouse blood vessels. Interestingly, the authors also found that MVs from the plasma of patients with atherosclerosis have high levels of miR-150 and that incubation of recipient HMEC-1 cells with these MVs replicated the effects seen (repressed c-Myb protein levels, increased cellular migration) when HMEC-1 cells were incubated with THP-1 MVs.

Several questions remain. As the evidence for selective packaging of miRNAs into MVs is tenuous, it remains to be determined if this is indeed true. If this is true, the mechanism of miRNA packaging would be a natural question to address and miRNAs that are processed differently might behave differently in this respect. In the immunology field, MVs are thought to be “zipcoded” by having different combinations of markers/receptors on their surface (Théry et al, 2002). This paper only tested HMEC-1 cells as the recipient cells and it would be interesting to see if monocytic MVs can be targeted to different cell types and thus modulate the cellular environment differently. In the paper, the delivered miR-150 appeared to repress c-Myb protein levels by ~4-fold, which seems rather high, even after taking into account that the c-Myb 3′UTR has two conserved 8mer seed matches to miR-150. It would have been nice if the authors had determined the concentration reached by miR-150 in the recipient cells, relative to endogenous miRNA concentrations, to see if this could explain the strong repression. Alternatively, as monocytic MVs (of a different cell line) were previously found to be enriched in GW182 (Gibbings et al, 2009), it would be interesting to see if this enrichment also occurs in THP-1 MVs and had somehow contributed to the strong repression observed. At the end of the paper, the authors suggest that finding high levels of miR-150 in the plasma MVs of atheroschlerotic patients may indicate that a contributing factor to atherosclerosis might be the secretion of MVs with high levels of miR-150 by stimulated macrophages, which then cause target endothelial cell migration. However, the cellular origin(s) of these plasma MVs was not determined. This hypothesis thus remains to be tested.

References:

Chen et al (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18: 997-1006

Gibbings et al (2009) Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat Cell Biol 11:1143-1149

Skog et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10: 1470-1476

Théry et al (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569-579

Valadi et al (2008) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654-659

Citation for researchblogging.org:

Zhang Y, Liu D, Chen X, Li J, Li L, Bian Z, Sun F, Lu J, Yin Y, Cai X, Sun Q, Wang K, Ba Y, Wang Q, Wang D, Yang J, Liu P, Xu T, Yan Q, Zhang J, Zen K, & Zhang CY (2010). Secreted monocytic miR-150 enhances targeted endothelial cell migration. Molecular cell, 39 (1), 133-44 PMID: 20603081

Target RNA–Directed Trimming and Tailing of Small Silencing RNAs

Stefan L. Ameres, Michael D. Horwich, Jui-Hung Hung, Jia Xu, Megha Ghildiyal, Zhiping Weng, Phillip D. Zamore

Science 328: 1534–1539, 18 June 2010.
DOI: 10.1126/science.1187058

This week’s thorough summary and analysis by Anonymous:

Like you, Drosophila siRNAs’d prefer an Argonaute; in fact their Argonaute of choice is Ago2. And this preference comes with a bonus: Hen1 adds 2′-O–methyl groups to all Ago2-bound small RNAs. In this paper, Ameres et al. attempted to uncover the mystery of the methyl group present in the 3’ end of Drosophila siRNAs, which is absent for most microRNAs. Adding an evolutionary twist to the story, in plants, all small RNAs (including microRNAs) are methylated. The obvious difference between the plant microRNAs and those of animals is the degree of complementarity to their targets. In plants, microRNAs are almost completely complementary to their target, hinting that the addition of the methyl group might be related to how tightly the small RNA is bound to its target.

Ameres et al. transfected several Drosophila cell lines with microRNA sensors, which had one or more fully complementary sites for an intended microRNA.  Normally these microRNAs are loaded to the Ago1; hence, they lack a methyl group in their 3’ end. They showed that levels of endogenously expressed (miR-34 and bantam) and induced (let-7 and miR-125) microRNAs decreased whence the corresponding microRNA sensor is present. Moreover, for another microRNA (miR-277), which can be loaded into both Ago1 and Ago2, they showed that the Ago1-loaded microRNA population is destabilized upon transfection of a microRNA sensor for miR-277. Therefore, Ago1-loaded small RNAs are prone to destabilization when a fully complimentary target is present.

In order to follow the fate of Ago1-loaded and destabilized microRNAs, the authors radiolabeled the 5’ end of let-7 with ³²P in the presence of let-7 sensor in vitro and observed new “tailed” and “trimmed” forms of let-7. They also detected tailed and trimmed forms of endogenously expressed bantam in the presence of a bantam-sensor using Drosophila embryo lysates. An addition of 3’ methyl group protected let-7 from being tailed and trimmed, and absence of Hen1 lead to tailing and trimming of let-7 siRNA. Putting all this evidence into context, the authors conclude that the methyl group protects small RNA from destabilization.

To determine the extent of tolerable complementarity between a microRNA and its target that does not lead to microRNA destabilization, the authors changed the sequence of complementarity site of the microRNA sensor. They found that targets resembling classical microRNA target sites did not result in tailing and trimming, whereas the targets that had less than 8 mismatches to the 3’ end of microRNA triggered destabilization. Furthermore, they also discovered that a small central bulge of 3nt leads to trimming and tailing, but not larger bulges. Hence, target RNA-triggered tailing and trimming require extensive but not necessarily perfect complementarity to the microRNAs.

Since Hen1 plays such a major role in protecting Ago2-bound small RNAs, the authors sequenced the small RNAs from hen1 mutant flies. The abundance and the length of microRNAs were not affected by lack of functional Hen1, but endo-siRNAs showed drastic changes in their lengths and abundance. The authors analyzed the content of the tails that arose due to absence of Hen1 and they found that the tail was either a single adenine or a single or multiple uridines, which is a mark of RNA turnover. Lastly, the authors also looked for target RNA-directed trimming and tailing in mammalian cells and confirmed the presence of a similar mechanism with slight modifications.

The Zamore Lab previously characterized RNA sorting in Drosophila, showing that the degree of complementarity between the small RNA and its star strand was responsible for being selectively loaded into Ago1 or Ago2. MicroRNAs are generally loaded into Ago1 to repress target translation and decrease target stability, often mediated by seed-paring between the microRNA and its target. The presence of highly complementary targets for Ago1 loaded small RNAs lead to remodeling of the small RNA by tailing or trimming. Therefore, Ago1 bound small RNAs are specialized in regulating partially complementary sites, which explain why microRNA targets lack extensive complementarity. The authors also speculate that extensive 3’ pairing might result in release of the small RNA from the PAZ domain of Argonaute, which in turn exposes the small RNA to the nucleotidyl transferases and 3’-to-5’ exonuclease enzymes.

Unfortunately we do not know which factors lead to target RNA-directed tailing and trimming and this will be an interesting venue of research in upcoming years. Moreover, the current model fails to explain the existence of highly complementary microRNA target sites present in both mammals and flies. But the paper formulates the question it tries to answer clearly from the beginning and delivers a satisfactory answer.

Citation for researchblogging.org:

Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z, & Zamore PD (2010). Target RNA-directed trimming and tailing of small silencing RNAs. Science, 328 (5985), 1534-9 PMID: 20558712

Down-Regulation of a Host MicroRNA by a Herpesvirus saimiri Noncoding RNA

Demián Cazalla, Therese Yario, Joan Steitz

Science Vol. 328: 1563 – 1566, 18 June 2010.
DOI: 10.1126/science.1187197

This week’s attentive summary and analysis by David Koppstein, his first on the blog:

In this study, Cazalla and colleagues found complementarity of three endogenous microRNAs–miR-27, miR-16, and miR-142-3p–to two noncoding RNAs encoded by Herpesvirus saimiri, HSUR 1 and HSUR 2, which have conserved motifs reminiscent of cellular U snRNAs. They pursued this observation by pulling down Ago2 and looking for these HSURs, and conversely by pulling down Sm proteins and looking for the miRNAs. Interestingly, these CoIPs specifically pulled down the HSURs and miRNAs, respectively, that were bioinformatically predicted to interact. During these experiments, they noticed that a mutant strain of the virus that lacked HSURs 1 and 2 expressed miR-27 at significantly higher levels.

Cazalla and colleagues then performed experiments to determine what was causing the increased levels of miR-27 in the mutant strain. They designed a pulse-chase nucleofection protocol with a radiolabeled synthetic miRNA, and noted a significantly shorter half-life in the presence of HSURs 1 and 2. Since levels of the pre-miRNA and the passenger strand were unchanged, they concluded that the mature miRNA itself must be destabilized.

It was also noted that the destabilization of miR-27 had consequences on the transcriptional landscape of the cell. A validated target of miR-27, FOXO1, which is a transcription factor that is dysregulated in breast, prostate, and endometrial cancers, was observed to be significantly downregulated in the absence of HSUR 1. Cazalla et al. also recapitulated the downregulation of miR-27 in Jurkat T-cells by
combinations of stable lines expressing HSURs and knockdowns using chimeric oligoribonucleotides. Strikingly, they observed that the specificity of HSUR 1 could be artificially switched to target miR-20.

There are several questions raised by this paper. First, there is the tantalizing prospect that the mechanism of destabilization of miR-27 is the same as that described by Ameres et al. in the same edition of Science. Further work, especially deep sequencing analysis of small RNAs, will likely reveal whether this is the case. There is also the mystery of where the interactions between HSURs and miRNAs are taking place; HSURs are thought to be nuclear, but may shuttle to the cytoplasm during their maturation. It is also still unclear what genes are perturbed as a consequence of the decreased miR-27 levels, and how this may affect viral fitness. Finally, one wonders about the functional significance of the interaction of HSURs 1 and 2 with miR-142-3p and miR-16. In summary, this paper presents a novel mechanism by which a virus perturbs host gene expression using noncoding RNA.

Citation for researchblogging.org:

Cazalla D, Yario T, & Steitz J (2010). Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA. Science, 328 (5985), 1563-6 PMID: 20558719

A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

Laura Poliseno, Leonardo Salmena, Jiangwen Zhang, Brett Carver, William J. Haveman & Pier Paolo Pandolfi

Nature 465: 1033–1038, 24 June 2010.
doi:10.1038/nature09144

No formal write-up for this week, rather just some points to consider, raised during our journal club discussion:

The authors’ probing of expression levels in prostate cancer cells showed that PTENP1 was expressed at much lower levels, perhaps up to 100-fold less, than PTEN. If this is true, how can CMV driven overexpression of PTENP1 RNA come close to recapitulating the relative levels and biological interactions of these two transcripts in the cell? The abundance of each transcript will strongly influence their abilities to act as miRNA sponges for one another. This concern wasn’t really alleviated even when they looked at expression in normal human and prostate tissue samples.

Since their central argument was that PTENP1 acts as a sponge for miRNAs that can also bind PTEN, since many miRNA binding sites are present in both transcripts, they were remiss in not showing the effect of expressing the PTENP1 3’UTR with mutated miRNA binding sites. This is a crucial control. While they showed that there is a difference in expression when the sites are mutated, using luciferase reporter constructs, the difference was small for the number of sites they predict are targeted by various miRNAs.

They also observed PTENP1 overexpression reduced colony formation significantly more than repressing PTEN protein alone, indicating expression of PTENP1 is important for reasons other than regulating how miRNAs bind PTEN (perhaps also not surprising given the near perfect maintenance of a large block of nucleotide sequence between the gene and pseudogene, not just the miRNA binding sites). Together, the data don’t provide adequate support to the authors’ claim that PTENP1 is an endogenous miRNA sponge for PTEN.

Update 7/8/10: A quick search for this paper on the web revealed it is quite popular with the ID folk–they’re eating it up good! And then I found the press release from Beth Israel Deaconess Medical Center, where Dr. Pandolfi says:

… not only have we discovered a new language for mRNA, but we have also translated the previously unknown language of up to 17,000 pseudogenes and at least 10,000 long non-coding (lnc) RNAs. Consequently, we now know the function of an estimated 30,000 new entities, offering a novel dimension by which cellular and tumor biology can be regulated, and effectively doubling the size of the functional genome.

Oh, well if you put it that way… now I see why it was in Nature.

Citation for researchblogging.org:

Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, & Pandolfi PP (2010). A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature, 465 (7301), 1033-8 PMID: 20577206

Update 9/8/15:

The Reproducibility Project: Cancer Biology is attempting to reproduce some results central to this study. Godspeed to you!

http://elifesciences.org/content/4/e08245