You'd Prefer An Argonaute

Keystone Nice

Posted in Conferences by YPAA on May 14, 2009

I attended the Keystone Symposia: The Biology of RNA Silencing meeting held in Victoria, British Columbia, Canada April 25-30th. The scientific organizers were Witold Filipowicz (who showcased his vigourous dance moves the last night of the conference), and Erik Sontheimer. I thought the meeting was very good, perhaps a bit better than last year’s. The format emphasis on having a greater number of shorter talks worked well generally, helping retain audience attention, although sometimes the quality of some of the workshop talks dipped a bit below my expectations. Below I will try to provide brief summaries of some of the scientific stories told in Victoria. (Sorry if details seem very disjointed, I’m going from notes far more than memory. And in general, I’m pretty careful taking notes, but mistakes happen, so I apologize if there are any errors.)

Numbers preceding speaker names correspond to abstract numbers.

It may will take me a few many weeks to get all the info up, so check back for updates.

Saturday

Keynote Session

001 Andrew Fire: “Adventures in the Small RNA-ome”

Fire opened the conference with the first keynote session. He started with a brief review of RNAi in C. elegans, noting that administering a pool of small RNAs that corresponds to only a few molecules per cell where they act is sufficient to completely knockdown a target mRNA that may be found at thousands of copies per cell. And that long dsRNAs can end up as small ~25nt RNAs. Of course this is through RdRP, of which C. elegans has 4 putative forms.

He then went into Sel-1 siRNAs. Some are cleaved products of the intial dsRNA trigger, of which sense and antisense are found equally. Antisense ones are also found mapping to regions outside the trigger. The majority have 5 prime triphosphates. Some span exon-exon junctions, indicating they are products of RdRP activity.

He then went into rrf-3 mutants, which have germline and somatic defects. These mutants have a decrease in expression of endogenous siRNAs.

Fire then went into data indicating a potential relationship between viral replication and vertebrate RNAi. Providing some context, he mentioned that some plant viruses encode proteins to block RNAi, suggesting that plant RNAi has an antiviral function. His lab is now looking for such a connection in vertebrates. Some preliminary results:

  • infection of HeLa with poliovirus results in many sense sRNAs from the viral genome.
  • infection of MEFs with poliovirus leads to a higher percentage of Ago2 loaded with sRNAs from the viral genome, changing the number of reads observed for several miRNAs
  • HepC virus infection of mammalian cell lines results in monophosphate vsRNA, although whether these have a role as pro-viral replication of anti-cell activity is not known. The vsRNAs confer very modest repression of reporters.
  • He ends stating that there remain strong arguments both supporting and against an antiviral role for mammalian RNAi.

002 David Bartel: “MicroRNAs and Other Small Regulatory RNA”

Bartel gave the second keynote. He opened with an update on computational results from analysis of miRNA target site conservation for 87 conserved vertebrate miRNAs that suggest that on average there are >480 sites per miRNA (from ~450 conserved targets), and that therefore >1/2 of human protein coding genes are miRNA targets. Among all these target sites, >90% only pair with the 5 prime end of the miRNA, or the ‘seed’. Another 7% of the conserved target sites exhibit 3 prime compensatory pairing.

For 54 mammalian specific miRNAs, analysis reveals 11 preferentially conserved sites per miRNA, mostly due to much higher background from a more limited species divergence.

Bartel then gave a most extreme example of a non-conserved target: a SNP that creates or destroys a miRNA target site in one allele of a gene in a single organism. He showed experimental evidence of how such SNPs can paritally explain differences in expression levels of the messages from the two alleles of such targets in vivo.

He then described a study of the interplay between alternative polyadenylation and transformation of cancer cells. In 6/16 genes studied, 1/3 to 1/2 of the differences in repression between short and long isoforms of transcripts (due to alternative polyadenylation) can be explained by the presence/absence of miRNA target sites. A correlation between transformation and shortening of 3 prime UTRs was shown, above that of proliferation and shortening, demonstrating the importance of miRNA target sites in the 3 prime UTR helping set appropriate expression levels of various oncogenes.

Bartel finished by describing the first discovery of RNAi in a budding yeast, Saccharomyces castellii . This yeast has a Argonaute, and a Dicer derived from a Rnt1 gene duplication. Its small RNAs are phased 23mers, mostly derived from TY and Y’ elements. While their function remains unknown, knockouts of Ago and Dcr have a plasmid maintenance defect. A robust reporter experiment showed the RNAi machinery in castellii can confer regulation of an integrated GFP reporter by a co-integrated hairpin harboring siRNAs antisense to the GFP transcript.

Sunday

Mechanism of Small RNAs I

003 V. Narry Kim: “Biogenesis of miRNAs”

Kim opened the first plenary session (which was moderated excellently by David C. Baulcombe). She talked about the let-7/lon-28 story. Her group had observed that let-7 was downregulated post-transcriptionally in mammalian cells. When they did an affinity purification for pre-let-7a and did mass spec, one of the hits was the protein lin-28. It has also previously been noted that in liver cancers and embryonic stem cells, lin-28b levels were low and let-7 levels high.

  • Lin-28’s regulation of let-7 happens mostly after Drosha action, with no effect on the hairpin up to the step of export
  • Lin-28 exhibits mostly cytoplasmic localization.
  • Gel shift experiments with lin-28 and pre-let-7 also produced a larger band corresponding to RNA above the pre-let-7 band.
  • Cloning of this band showed it was a uridylated pre-let-7, with U tails of an average length of ~14 nts.
  • The poly-U activity was dependant on WT lin-28 protein.
  • uridylated pre-let-7 is resistant to dicing (undicible), and decays faster than pre-let-7

Another hit was the original mass spec experiment was Tut4, containing a zinc-finger and uridylation catalysis motif.

  • knocking down tut4 with siRNA leads to an increase in mature let-7 levels without any change to pri-let-7 levels
  • targeting lin-28 and tut4 independently with siRNAs followed by a microarray shows an increase in levels of 5 members of the let-7 family, but no change in any other miRNAs
  • her group managed to do in vitro uridylation experiments, and here only WT tut4 could mediate uridylation of pre-let-7, but not any other pre-miRNAs tested.
  • tut4 activity is lin-28 dependent, and tut4’s interaction with pre-let-7 is lin-28 dependent. To summarize: lin-28 is required for tut4 recognition of pre-let-7, tut4 requires pre-let-7 to interact with lin-28, and therefore tut4 recognizes a binary complex
  • it isn’t known if there are other tut4 like homologs in worms, or whether the uridylation is reversible.

It is also worth noting that plant mature miRNAs are uridylated, although these tails are on average much shorter than 14 nts.

004 Gunter Meister: “Regulation of Argonaute Function”

Meister talked about a snoRNA that may act like a miRNA and regulation of Argonaute function. He stated that he now has antibodies that can discriminate between the 4 human Ago proteins.

  • > %90 of sRNAs bound to Ago 1 and 2 are miRNAs, but he also found some snoRNAs bound as well.
  • snoRNA ACA45 is processed to a conserved sRNA. snoRNPs fall into 2 classes with different core proteins, and antibodies can recognize snoRNP proteins to precipiate snoRNAs, like ACA45.
  • in vitro, ACA45 is processed in a Drosha/DGCR8 independent, Dicer dependent manner. Recomninant dicer also works.
  • in vivo in a Dcr knockout, full-length ACA45 is present, but the sRNA derived from it absent
  • the sRNA products derived from ACA45 only derive from the 3 prime end of the snoRNA, a conserved sequence in mammals.
  • his group reports finding one 3 prime UTR regulated by the snoRNA (I think using reporter assays.)

A model for these snoRNA sRNAs is that for a given snoRNA with active function in the nucleus, some fraction of it is transported, diced, and may regulate a UTR, probably in the cytoplasm. Other snoRNA are also predicted to be processed to sRNAs.

His group has also seen sRNAs derived from full-length tRNAs in Ago precipitates. Fragments of these can guide cleavage of a perfectly complementary target RNA in vitro. (YPAA (that’s me!) found this result quite dubious and asks what RNA fragment cannot guide such cleavage of a perfect target in vitro? Also he gave no mention of targets in the genome, conservation, etc.) He finds 10-26 nt fragments of this tRNA bind Ago 2, guiding cleavage. “Ongoing work.”

Then as I pondered the uselessness of this last result, he got to the most interesting part of the talk, regulation of Ago function, and I was asleep at the wheel! So sorry for not getting much here.

  • looking for Ago2 peptides that are phosphoryated by using mass spec
  • Ago2 Y529E mutant doesn’t cleave target RNA in vitro
  • this mutation results in a strong reduction in the proteins interaction with miRNAs (I believe the 529 position is in the 5 prime binding pocket, but I’m not sure on this.)
  • In summary, phosphorylation of Tyr 529 in Ago2 may act as a switch to inactivate Ago2 activity for binding sRNAs and directing cleavage.
  • Cool stuff indeed!

229 Qinghua Liu: “C3PO Promotes Drosophila RNAi by Enhancing RISC Activity”

Liu talked about RISC composition, assembly, and regulation in Drosophila RNAi. Defining a core RISC as dicer2/R2D2/Ago2, he set out to find factors that enhance RNAi. He talked about a novel activator of RISC activity, named C3PO. (Indicating Liu is a Star Wars nerd.) I neglected to take down too many details of C3PO, but here’s what I got:

  • Proteins Traslin and Trax members of C3PO, both have mostly unknown functions in other contexts
  • Traslin is a ssRNase that enhances passenger strand cleavage, implying its main function is to facilitate removel of cleavage products
  • both proteins are required for efficient RNAi in vivo
  • recombinant proteins can also promote RISC activity

274 Bruce Paterson: “Identification of an RNA-Dependent RNA Polymerase in Drosophila Involved in RNAi and Transposon Suppression”

Ok so my notes for Bruce’s talk are a little muddled, so I apologize, but here’s what I got:

He began by describing how generally RdRPs in the fly have a role in RNAi and transposon suppression. He then introduced that D-elp-1 has RdRP activity. This is true for the recombinant version, whether introduced by a virus or purified from E. coli. And that indeed D-elp-1 appears to have role in dsRNA silencing as well as transposon suppression. In a D-elp-1 null or depleted (RNAi’d) line, there are changes in abundance of sense and antisense RNAs to a target gene. D-elp-1 appears to be cytoplasmic and associated with Dcr2. It appears to function in a “non-canonical, alternative RdRP pathway”, as he described it. D-elp-1 contains no domains that identify it as a strong candidate to be a polymerase, and therefore it wasn’t seen earlier.

005 Erik Sontheimer: “CRISPR Interference in Bacteria”

  • CRISPRs are composed of repeats and spacers, generating RNAs that often match phage genomes
  • repeats + spacers = “pre-crRNA”
  • crRNAs (mature forms) block phage infection very robustly, mismatches at certain positions between RNA and phage block this function
  • CRISPRs are adaptive, bacteria can add novel sequences to the repeats/spacers
  • Staph epidermis harbors a CRISPR locus
  • deletion of a CRISPR locus can be rescued with a plasmid expressed version
  • CRISPR interference targets DNA, so how to distinguish self vs. non-self? Q: Are CRISPR spacers targets for interference? A: No. When spacers themselves are transformed in, there is no response.
  • When one starts to remove repeats, can see interference from self-targeting, lose protection, is position dependent
  • Perfect pairing of crRNA with encoding DNA blocks interference — mismatch at position 2 required for interference between crRNA and target DNA
  • A candidate effector protein for CRISPR interference has an ATP helicase and novel nuclease, but the mechanism remains unknown

006 Dinshaw Patel: “Structural Biology of Argonaute-Mediated RNA Silencing”

Let me preface this summary by saying that Dinshaw consistently gives an excellent talk at Keystone. He always presents exciting new data, and his presentations are crystal clear, great slides, etc.

Dinshaw discussed the Thermus thermophilus Argonaute structure, which contains N, PAZ, MID, and PIWI domains. The first set of bullet points concern the binary Ago-guide strand complex, the second set a ternary Ago-guide-target complex

  • for an integrated guide strand DNA, nucleotides 1-12 and 18-24 are traceable in the structure
  • the 5 prime phosphate hydrogen bonds with conserved residues in the MID pocket, and the 2-nt 3 prime end rests in the PAZ domain
  • recognition of the 5 prime phosphate is more important than recognition of the 3 prime end for cleavage
  • bases 2-10 of the guide strand are stacked even in the absence of a target oligo, and the guide strand can hydrogen bond with the protein through contacts with its sugar phosphate backbone
  • stacking of guide strand bases is disrupted by an Arginine at the 10-11 step of the structure
  • bases 2-6 of the guide have their Watson-Crick edges pointed outward from the protein, allowing nucleation with target, the rest of the bases “enter” into Ago
  • in a non-cleavable ternary, seed positions 2-9 can be seen, but no nts beyond position 9
  • the sugar-phosphate backbone of the target is not predicted to interact with Ago
  • the Patel group solved a number of ternary complex structures with a Ago mutant (at catalytic site) with various length target oligos: 12, 15, and 19mer
  • in the guide strand, only the first base is recognized specifically, the backbone is recognized up through base 10
  • there are large shifts in the complex structure in going from the the binary to ternary structures
  • in moving from the 12mer target ternary to the 15mer ternary, the 3 prime end of the target is released from the PAZ domain
  • Ago can accommodate a maximum of 15 bps (positions 2-16) of guide strand-target duplex (implying any predicted potential pairing beyond position 16 is inconsequential)
  • Modifications of a 2 prime hydroxyl at/near the cleavage site between positons 10-11 had various consequences depending on the nature of the mutation: an F increased cleavage, a 2 prime – O – methyl decreased cleavage, a deoxy had an intermediate change
  • modification of the cleavage Phosphate also yields changes in efficiency
  • bulges in the guide strand seed don’t totally impair cleavage, but can alter the position of cleavage
  • a single bulge in the target strand caused a U to loop out, and an unexpected A:A bp that displaced the cleavage Phosphate and shifted the cleavage position by one phosphate

While summarizing, Dinshaw said Ago domains pivot relative to eachother in binding through nucleation, propagation, and cleavage (can change at each step). Ago can undergo dramatic structural changes, fairly independently of duplex sequence

233 Jin-Biao Ma: “Structural Insights into the Molecular Mechanism of the Small RNA Methyltransferase HEN1”

  • the 3 prime end of sRNAs can be methylated: plants, piRNAs, siRNAs in fly, others
  • HEN1 is well conserved
  • the substrate for methylation is the 2 prime OH on the terminal nucleotide, and methylation requires duplex RNA
  • HEN1 uses multiple domains to recognize the dsRNA substrate, including optimal length of the dsRNA

Monday

Mechanism of Small RNAs II

010 Scott Kennedy: “siRNAs Initiate a NRDE-2-Dependent Co-Transcriptional Gene Silencing Program that Regulates RNAP II Elongation

“How and why do sRNAs function in the nucleus?” Kennedy asks to begin his talk. For nuclear RNAi, if you gene A and gene B are in cis, and you interfere with gene A, silencing it, will gene B also be silenced? Yes, he says, as targeting an upstream gene of an operon will also silence the downstream gene. So to carry out a screen for factors involved in nuclear RNAi, his group screens for mutants where in the above example the downstream genes (“gene B”) is not silenced.

  • NRDE 1-7 known
  • Nrde-3 encodes an Ago like protein required for silencing in nuclear RNAi, and its NLS is also required for silencing; it is normally present in nucleus, and must arrive there to initiate RNAi. Is it an siRNA shuttle?
  • ERI/DCR required for endogenous siRNA production/accumulation
  • siRNA binding is necessary and sufficient to direct NRDE3 to the nucleus
  • NRDE-2 is also required for nuclear RNAi, is a conserved protein of unknown function, acts downstream of NRDE-3, and localizes to the nucleus in an siRNA independent manner, and associates with the NRDE-3 protein
  • Kennedy’s model is that NRDE-3 associates with siRNAs in the cytoplasm, and then is directed into the nucleus where it is recruited to nascent transcripts by NRDE-2 where it directs HSK9 methylation. (my notes are totally clear here, it appears NRDE-2 goes to nascent transcripts in an NRDE-3 dependent manner but I’m not sure which NRDE is doing the methylation)
  • RNAi triggers loss of pre-mRNA 3 prime of the RNAi site
  • NRDE-3 associates with RNA fragments 5 prime to the site of RNAi
  • slicer activity is not required, and NRDE-3 doesn’t have possess any anyways
  • there’s an observed increase in RNA pol II occupancy where RNAi acts
  • Kennedy drew a rough possible model where N3 binds nascent transcripts through siRNAs, N2 is associated with this and somehow blocks Pol II activity just downstream, possibly inhibiting elongation, but the true mechanism of silencing remains unknown

011 Craig Mello: “Complexity of RNA Silencing in C. elegans

  • RDE1 is the only Ago absolutely required for viability
  • WAGOs (including NRDEs) can be completely deleted and viable
  • an uncharacterized Ago is involved in chromosome segregation
  • kinetochores are disorganized in csr-1 mutants
  • DRH-3 (Dr related helicase) is required for 22-G RNA biogenesis
  • ends of 22-G RNAs are tri-Phosphorylated, RNAs are RdRP products
  • 22-Gs are antisense to 60-70% genes, including genes required for fertility and embryogenesis
  • 22-Gs engage distinct germline Agos
  • csr-1 and WAGO-1 interact with a non-overlapping set of 22-Gs
  • 22-Gs engaged by csr-1 include those antisense to germline expressed coding genes
  • only the most abundant 22Gs appear to alter mRNA levels

012 David C. Baulcombe: “Imprinted Expression of siRNA loci in Arabidopsis”

I have very limited, and potentially confusing, notes for this talk, my apologies:

Pol IV and V are variant forms of Pol II. Pol IV copies transposable elements, repetitive sequence, very few genes; can make dsRNA of these templates through RdRP. These dsRNAs can be cleaved by Dcr3 into 24 nt RNAs. These RNAs can associate with Ago4, and Pol V transcripts to modify DNA. There are Pol IV and Pol V dependent loci throughout chromosomes, especially at centromeres. Pol IV siRNAs are imprinted.

013 Peter Sarnow: “Modulation of Hepatitis C Virus RNA Abundance by Liver-Specific microRNA miR-122

  • miR-122 makes up 72% of liver miRNAs, some cultured liver cells express it and some do not, expression levels go down upon transfection, and the expression levels of the miRNA are correlated with the ability of Hep C virus to replicate in the cells
  • only poor anti-HCV compounds exist now
  • Hep virus has 1 site for miR-122 in its 3 prime UTR, and two sites in the 5 prime UTR, these seed match sites are highly conserved, and the 50 nt spacer for the dual sites in the 5 prime UTR has an absolutely conserved length, (but its sequence is less important). [Yes this is not a typo – despite HCV’s target sites for miR-122, the miRNA appears to somehow facilitate HCV replication.]
  • sequestration of miR-122 with antagomirs lowers intracellular abundance of HCV RNA. Introduction of a corresponding mutated miR-122 mimic for site 1 can rescue lack of espression of HCV, also works for site 2 targeting
  • genetic evidence shows both sites are required for rescue
  • mutation of seed sites does not affect translation of HCV RNA
  • miR-122 does not affect replication of HCV
  • Sarnow group used thio-uridine tagging of newly synthesized RNAs to investigate localization
  • miR-122 is associated with non-polysomal and polysomal factors, so multiple inhibition of different steps?
  • ectopic expression of miR-122 enhances HCV RNA abundance
  • with staining, 122 localizes to discrete subcellular structures, not sure if they are P-bodies, however P-bodies do disperse after infection with HCV
  • some factor (I wrote down “RCK”, I have no recollection of what this is) in P-bodies may be important for miRNAs to positively regulate viral RNAs, in this example 122 controlling HCV RNA abundance at a mostly post-transcriptional step. RCK has positive effects on the HCV replication cycle
  • Sarnow got a great question where someone asked if you transfect a reporter with the 3 prime UTR 122 sites, then infect cells with HCV, what is the effect on the reporter? He hasn’t done the experiment yet.

Yukihide Tomari: “Dissection of Ago1-RISC Assembly Pathway in Drosophila

  • How are miRNA duplexes loaded into Ago1?
  • Tomari defines a passive process (does not require ATP or slicer activity) where “pre-Ago RISC” binds the duplex, and through unwinding, leads to formation of the “mature Ago-RISC” bound with ssRNA.
  • central mismatches direct Ago1-RISC loading, in contrast to Ago2 loading which prefers perfect match duplexes
  • mismatches in the seed and 3 prime end also promote unwinding
  • *Duplex unwinding is the mirror image of target recognition*
  • GU wobbles behave like mismatches for unwinding
  • natural miRNA/miRNA* duplexes are suitable for RISC loading and unwinding – example: many fly miRNA duplexes have central mismatches

Eric C. Lai: “Sorting of miRNA* Strands in Drosophila

  • Lai reiterated the “Loading Rule of Strand Selection” in flies: bulges/mismatches in miRNA duplexes lead them to Ago1 loading whereas perfect matches favor Ago2 loading, but then added more about endogenous miRNA* targeting
  • some miRNA* species regulate endogenous targets via Ago1
  • most miRNA*s only regulate perfect match targets, through Ago2 loading (IP evidence shows preference for loading *s into Ago2)
  • 2/3 *s have preferred incorporation into Ago2, above the ratio in total RNA
  • in vitro assay supports prevalent loading of *s into Ago2
  • bulges may override thermodynamic end rule of loading, so central pairing a critical determinant of loading preference?
  • Lai proposes two potential reasons why *s are loaded into Ago2: (1) to draw * away from Ago1 (2) there are endogenous targets for miRNA*s

YPAA thinks these last two hypothesis are poorly supported at present. For (1), a miRNA will be loaded into Ago1, and the pasenger strands will be discarded; it is of no concern to the miRNAs that they have these partner strands floating around, because they will loaded into Ago1 presumably at very low frequency. And how does it help loading of miRNAs into Ago1 by loading *s into Ago2? If *s get loaded into Ago2, their passenger strands, miRNAs in this case, will be discarded as in the reciprocal case. So that doesn’t help either. For (2), Lai didn’t present any evidence (at least that I wrote down – I try to write down all the main points presented by each speaker) for endogenous targets of these *s. It is so easy to look, I’m sure that his group has looked. So I’m not sure what to make of these results. (Note: Like most/all of you, I get paid to be skeptical.)

Workshop I: Identification of New miRNA Targets

40 Mihaela Zavolan: “Sequence and Structure Determinants of miRNA Targeting Specificity”

To put her group’s data in context, Zavolan started by mentioning that in miRNA overexpression experiments, the RNAs primarily lead to mRNA target destabilization, in contrast to translational repression, and that conserved seed matches are more effective than non-conserved ones. Given these facts and others, her group set out to quantify selection strength on miRNA target sites using a large number of previously published data sets as well as an in house HEK293 transfection with miRNAs.

  • algorithm EIMMO
  • found several parameters affecting selection strength: structural accessibility, AU and CG content in the target site “environment”, or in the transcript itself.
  • found 2 feedback loops: one involving let-7 targeting Dcr; and another involving miR-30 targeting TNR6C
  • structural features only effect binding efficiency, not degradation
  • translational inhibition is the “exception rather than the rule”
  • “structural features only tell about RISC binding and little about whether an mRNA will be degraded or not”

163 Artemis G. Hatzigeorgiou: “DIANA-MicroT 4: Machine Learning Based microRNA Target Prediction”

This talk was incredibly dense and unavailing. I’ve been told this algorithm performs fine, but the talk was terrible. My short, jumbled notes reflect this.

  • artificial recurrent neural network trained on SILAC data
  • looked at structural accessibility on a window of 300 surrounding nucleotides to target site
  • protein folding change scores used for training set
  • DIANA is as precise, but more sensitive than Targetscan or PicTar

161 Markus Hafner: “PURE-CLIP – Transcriptome-Wide Identification of miRNA Targets and RNA Targets and Binding Sites of RNA-Binding Proteins”

Word on the street is this work will be coming out soon. (Perhaps a Cell Resource paper?)

The method summarized:

  • Feed mammalian cells 4-thio-U nucleosides, are incorporated into new transcripts.
  • Crosslink 4-thio-U to the aromatic side chains of residues in RNA binding proteins (RBPs) using long-wavelength UV irradiation.
  • lyse cells, IP RBP, trim RNA to get purified fragments, end label, run on PAGE gel, do high-throughput sequencing
  • align sequence reads, identify clusters, see where distribution lies

Hafner’s notes in the abstract:

Importantly, the position of the crosslink and therefore the true RNA recognition site of the protein can be deduced from the characteristic T to C transition in the reverse transcribed cDNA.

Apparently this phenomenon improves the signal to noise for bound regions.

  • > 50% of ~12,000 identified miRNA target sites found in the CDS of this Ago-PURE CLIP method
  • in the abstract it is stated: Inhibition of the top expressed miRNAs and analysis of mRNA abundance showed that these binding sites are functional and that miRNA binding leads to mRNA destabilization.
  • seed complements are the most abundant 7mers recovered
  • 20% of transcripts are targets
  • 80% sites have at least one 6mer, other 20% enriched for more GU wobble mismatches
  • for the 50% of sites found in the CDS, these sites are surrounded by unconventional codon usage
  • rank of destabilization levels by site type: 6mer < 7mer < 8mer < 9mer seed match

371 Dimitrios G. Zisoulis: “Massive Identification of miRNA Target Sites in Endogenous mRNAs”

This work comes from Amy Pasquinelli’s lab, and they did a CLIP-seq on ALG-1 from C. elegans to look for miRNA target sites.

  • Background defined by sites recovered from an ALG-1 mutant (he told me that nature of the mutation was an early stop codon leading to what he said was a shortened, non-functional peptide). Binding sites overlapping between WT and ALG-1 mutant are eliminated. I believe the worms were all L4.
  • method validated a let-7 site in lin-41; and several sites for lsy-6 including cog-1 and a couple others (he did not say where in the UTRs the reads mapped to, in other words, if the reads overlapped predicted seed matches, or just somewhere near them. This is one significant drawback of this method to predict or validate miRNA target sites, you don’t know which miRNA was loaded into Ago when it was attached to a given UTR site. So this will be very nice when they do CLIP-seq on miRNA knockouts.)
  • recovered general CU-rich motifs
  • enriched for seed pairing, however only 15% of sites have conserved seed matches
  • only 2% of sites were predicted by all 6 target prediction programs used (Hmm, either a lot of these sites are bogus, or the overlap between 6 of the most highly used T.P. algorithms is ultra-low, or a combination of both factors.)

250 Francesca Moretti: “Positional Effects of microRNA-Mediated Translational Regulation and Their Mechanistic Basis”

This work comes from the lab of Matthias W. Hentze. Using an in vitro system composed of Drosophila cell-free embryo extracts, Moretti investigated regulation of reporters for mir-2 to examine the effect of introducing target sites in the 5 prime UTR or ORF. A 6X tandem mir-2 target site chunk can be repressed when located in the 5 prime UTR, the beginning or end of the ORF, and the 3 prime UTR, and an anti-mir-2 LNA can abolish repression for all these cases. A single mir-2 site is only effective in the 3 prime UTR. A double mir-2 site chunk is effective in the 5 prime UTR as well as 3 prime UTR.

She concludes that the mechanism of repression of these reporters is inhibition of translation initiation, based on data showing inhibition of 80S ribosome assembly and formation of pseudo-ribosomes in the ORF and UTRs without mRNA destabilization.

Intersection of RNAi/miRNA Silencing with Other Cellular Pathways

014 Olivier Voinnet: “RNAi and Pathogen Defense in Plants”

My notes for Olivier are quite meager:

  • many viruses target Dcr4 or its products
  • P38 binds to Ago1 through GW182 to block Ago activity
  • P38 binds to MID/PIWI domain, an example of molecular mimicry

015 Bryan R. Cullen: “Viruses, microRNAs and RNA Interference”

  • no support currently for viral induction of an antiviral siRNA response in mammalian cells
  • virus encoded miRNAs could help regulate themselves
  • lots of Herpes viruses have miRNAs, none found in RNA viruses
  • virus would sacrifice its genome to make a miRNA due to biogenesis processing steps
  • makes more sense kinetically for viruses to target protein production rather than mRNA abundance

An example: KSHV

  • viral miRNAs resemble other miRNAs
  • miR-155 shares seed and targets with cellular miR 12-11, similar effects on predicted targets, functions as a mimic with oncogenic potential
  • Epstein-Bar virus doesn’t have miR-155, but it does activate expression of endogenous miR-155
  • overexpression of these miRs can lead to formation of lymphoid tumors

HSV:

  • causes latent infection, no good cell culture model exists
  • in its latent stage, only expresses a single ncRNA, LAT
  • a perfect complementary target to LAT is downregulated without significant degradation (mRNA?)
  • identified 7 new HSV-1 miRNAs, some regulate viral mRNAs
  • LAT may act to favor entry into latency
  • exonic LAT transcript is unstable b/c it codes miRNAs
  • Q: Why make an unstable ncRNA? — A: miRNA expression can promote latency

016 Craig P. Hunter: “Intercellular RNA Transporters in Worms and Vertebrates”

  • made SID mutants using double GFP reporter strain that monitors transport/spread of RNAi
  • SID-2 is a lumen protein highly variable between closely related species, intracellular part is well conserved

SID-1

  • whole cell patched S2 cells expressing SID-1, a dsRNA gated channel
  • if you trypsinize cells, can get concentration of dsRNA inside equal to outside
  • import/export is rapid process, energy independant
  • miRNA precursors can go through; dsDNA is not transported, therefore structure matters
  • summary: SID-1 is a dsRNA gated channel, not size selective, specific to RNA (not DNA)

SID-2

  • SID-2 mutants are defective for environmental RNAi
  • transport only works at pH=5 or lower, like lumenal uptake of dsRNA
  • slow uptake, endocytosis
  • evidence of transcytosis of SID-2, re-entry of dsRNA through SID-1 –> spread of RNA
  • knockdown of vertebrate SidT1, T2 causes embryonic lethality, cell growth, cell cycle defects
  • in frogs and mice, expression higher in proliferating cells
  • Sid 2 knockdown (knockout?) mice are small, reduced MEF growth
  • in frogs, chromosome segregation and cell cycle defects

Yi Liu: “A Novel Type of Small Interference RNA Induced by DNA Damage”

This was an interesting talk, and the work was published a couple weeks after the meeting ended so I’ll refer you the the primary paper.

more details to come for the following speakers…

Tuesday

Regulation of Translation and Stability by miRNAs

017 Elisa Izaurralde: “GW182 Proteins are Essential for miRNA-Mediated Gene Silencing in Animal Cells”

018 Witold Filipowicz: “Mechanism and Regulation of miRNA Repression in Mammalian Cells”

019 Nahum Sonenberg: “Dissecting microRNA Mechanisms of Action in vitro

020 Joan A. Steitz: “MicroRNPs: Versatile Regulators of Translation in Vertebrate Cells”

159 Helge GroBhans: “Active Turnover Modulates Mature microRNA Activity in C. elegans

Nikolaus Rajewsky: “Dynamic Small RNA Expression in Precisely Staged Early C. elegans Embryos and the Planarian Small RNAome”

302 Priyamvada Rajasethupathy: “Characterization of Small RNAs in Aplysia Reveals Neuronal piRNAs and a Neuron-Specific miRNA that Constrains Long-Term Synaptic Plasticity”

A long name, and a very long title!

244 Michael Thomas McManus: “EXPANDed RNAi Libraries for High-Throughput Deep Screening”

RNA Silencing in the Germline

027 Rene Ketting: “RNAi and Chromosome Segregation”

Wednesday

MicroRNAs in Development

028 Richard W. Carthew: “Silencing by Small RNAs is Linked to Endosome Trafficking”

This was one of the most interesting talks at the meeting, a fresh new study I heard may come out in Nature Cell Biology soon.

  • Ago proteins associate with cytosolic membranes
  • to be continued….

029 Victor Ambros: “MicroRNA Pathways in Animal Development”

176 Hun-Way Hwang: “Cell-Cell Contact Globally Activates microRNA Biogenesis”

364 Yang Yu: “Enhanced Magnitude of miRNA-Mediated Regulation Upon Muscle Differentiation”

257 Joel Neilson: “3′ UTR Dynamics in Cellular Activation and Transformation”

Small RNAs in Medicine

Sorry but I have no notes from any speakers from this last, final session of the meeting! I can say that Tariq M. Rana gave an *excellent* talk entitled “Role of P-Bodies and MicroRNAs in Modulating Host-HIV-1 Interactions.” Look for this work in the literature! (Just came out in June 26 issue of Molecular Cell I think.)

FIN



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6 Responses

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  1. Yang said, on May 15, 2009 at 2:59 pm

    Hi,
    Do you know why they don’t look at coding regions for microRNA targets? Do they have any evidence that nothing is happening in the protein coding regions?

    • YPAA said, on May 15, 2009 at 6:26 pm

      Hello,
      Great question. In animals, researchers have found some evidence for functional miRNA targeting in the open reading frame (ORF), but is has never been shown to be as strong as that found for sites in the 3 prime UTR. [It is worth noting that in plants the target sites, which are often perfectly complementary to the full mature miRNA sequence, are more often found in the ORF than in the 3 prime UTR (Jones-Rhoades and Bartel 2004).]

      In animals several lines of evidence suggest that the reason for this is that translation of the ribosome through the ORF impedes association of a miRNA loaded RISC with the mRNA (see Grimson et al. 2007 and Gu et al. 2009).

      Two studies utilizing genome-scale proteomics methods to study the effects of miRNA targeting demonstrated a very modest effect of seed matches in the ORF on protein output (Baek et al. 2008 and Selbach et al. 2008).

      From a computational standpoint, predicting functional miRNA target sites by their conservation in the ORF is confounded by the fact that most nucleotide content in the ORF of a conserved gene is, well, conserved. So the background is much higher compared to UTRs (Lewis et al. 2005). Nonetheless, you can find many seed matches in the ORF, some conserved above background, but current experimental evidence suggests they have only a weak effect on protein expression.

      • Yang said, on May 17, 2009 at 4:13 pm

        Thanks a lot for the references! The reason I was asking this is that in a soon-to-be-submitted paper(probably this week), we devised a way to decouple the amino acid background from the “real” conservation signal to search for ultra-conserved DNA elements, we found that many of the exact matches of the animal miRNA seed sequences from the Friedman et al. 2009 paper actually have very high conservation scores. We suspect this probably mean something…

      • Yang said, on May 17, 2009 at 4:15 pm

        Oh I missed a keyword: we devised a way to decouple the amino acid background from the “real” conservation signal to search for ultra-conserved DNA elements in “protein coding regions”…

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