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Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans

Sarah E. Calvoa, David J. Pagliarinia and Vamsi K. Mootha

PNAS 106 (18): 7507-7512, May 2009.
doi: 10.1073/pnas.0810916106

This week’s incisive summary and analysis by Robin Friedman:

Upstream ORFs (uORFs) generally consist of an AUG codon with an in-frame stop codon preceding the end of the canonical coding sequence (CDS).  The uORFs therefore can either be entirely upstream of the CDS or overlapping the start of the CDS.  uORFs have been shown to decrease CDS expression in many anecdotal cases, although translation of the CDS can still occur by leaky scanning or re-initiation.  Early analysis suggested that <10% of vertebrate mRNAs had upstream AUGs, but more recent computational predictions suggested that >40% of vertebrate genes have uORFs.  This study is the first to experimentally address the extent of uORF impact on a genome-wide scale.

The authors constructed a 5′ UTR dataset from refgene annotations, finding that 49% of human and 44% of mouse transcripts have at least one uORF.  They next examined high-throughput MS/MS datasets for steady-state protein quantification at a genome-wide level.  In each of four datasets, genes that have uORFs have lower protein expression than genes with no uORFs, even after normalizing to mRNA expression.  uAUG context, the distance from cap to uORF, uORF conservation, and the number of uORFs all affected this difference in protein expression, whereas uORF length and distance from uORF to CDS did not.

While the previous experiments show that uORF-containing genes have lower steady-state protein levels, they do not show a direct effect of uORFs on translation.  To test directly whether the uORFs affect translation, the authors created reporters with the 5’UTRs from randomly selected genes containing uORFs fused to luciferase.  Compared to a single-nucleotide-mutant that removes the uAUG, the luciferase activity was reduced ~50% in five randomly selected mouse genes, while the mRNA level, assayed by qPCR, was mostly unchanged.  For 10 mouse genes with MS/MS and conservation support for functional uORFs, the luciferase reporters showed 50-80% repression at the protein level.

Asking whether the uORFs could be involved in human polymorphism and disease, the authors queried dbSNP and the human gene mutation database for mutations that create or destroy uORFs.  There are 509 genes with polymorphic uORFs, and 14 with recorded mutations linked with disease.  Five of the polymorphisms were tested by qPCR, and the uORF was found to repress protein levels by 30-60%, while five of the disease-causing uORF mutations were found to repress by 70-100%.

This paper convincingly argues that uORFs are widespread in humans and have a widespread impact on protein expression.  Much of this impact is likely conserved and functional.  In addition, they provide interesting experimental support for the fact that uORFs typically repress at the translational level as opposed to through NMD and that CDS translation downstream of uORFs likely proceeds from leaky scanning rather than from re-initiation.  While the mechanism has not been elucidated on a genome-wide scale, this paper provides an refreshing look at an often-ignored but important contribution to translational control.

A Role for RNAi in the Selective Correction of DNA Methylation Defects

Felipe Karam Teixeira, Fabiana Heredia, Alexis Sarazin, François Roudier, Martine Boccara, Constance Ciaudo, Corinne Cruaud, Julie Poulain, Maria Berdasco, Mario F. Fraga, Olivier Voinnet, Patrick Wincker, Manel Esteller, Vincent Colot

Science 323 (5921): 1600-1604, March 2009.
doi: 10.1126/science.1165313

This week’s summary and expert analysis by Michael Nodine:

DNA methylation of transposable elements occurs through both RNAi-dependent and RNAi-independent mechanisms in plants. Methylation of transposable elements leads to their silencing and maintains genomic stability. Mutations in methylation components, such as the maintenance methyltransferase MET1 and the chromatin remodeler DDM1, lead to a loss of >70% of genomic methylation. The progeny from met1 x wild-type (WT) and ddm1 x WT crosses have reduced methylation despite these mutations being recessive. Furthermore, when these heterozygous plants are selfed and the MET1 and DDM1 loci are restored to the homozygous WT condition, several loci remain hypomethylated. Based on these findings, it has been proposed that once methylation is severely compromised it cannot be restored and thus is permanently lost. However, comparisons between different Arabidopsis accessions revealed that the methylation patterns of repetitive elements were similar across generations. This suggests that a mechanism exists to prevent permanent loss of DNA methylation. That is, there must be a way to specifically and robustly reestablish methylation.

In this study, Teixeira et al. set out to identify the mechanism that underlies this methylation reestablishment. First, they crossed the methylation defective mutant ddm1 with WT, recovered DDM1 F2 plants and selfed these plants for several generations. They then examined the methylation levels of several loci in the heterochromatic knob region of chromosome 4, and found that methylation was restored for ~50% of the repetitive loci examined (remethylatable sequences (R)), but not for the other ~50% (non-remethylatable sequences (NR)). The patterns of NR and R sequences were consistent between different independent lines. Remethylation did not occur in the F1 generation, but was progressive from the F2 generation onwards and led to silencing of transposable elements. In contrasts to previous models, these findings indicate that a robust and targeted remethylation process takes place.

The authors went on to demonstrate that cytosine remethylation occurred in all three sequence contexts (CG, CHG and CHH where H it A, T or C). They also found that NR sequences had stronger dependence on DDM1 for CHH methylation than did R sequences. Since RNAi components have important roles in CHH methylation, this observation led to the hypothesis that RNAi may be involved in remethylation of R sequences. To test this, they examined small RNA datasets and found that R sequences had a strong association with small RNAs especially 24-nt heterochromatic siRNAs (hc-siRNAs), which are involved in RNA-directed DNA methylation. Moreover, when they combined mutations in the RNAi machinery with ddm1 they observed an enhanced loss of methylation at both R and NR sequences suggesting that RNAi plays a role in the methylation at both types of loci. However, mutations in RNAi components (when not combined with ddm1) resulted in decreased methylation at R, but not NR, sequences. To demonstrate that RNAi plays a direct role in remethylation, they examined whether R sequences were remethylated when RNAi was compromised in the initial generation and found that only sporadic and inconsistent remethylation occurred in several independent progeny lines. Together, these results indicate that RNAi is involved in robust remethylation at specific loci.

Based on these findings, the authors propose that there are three types of methylated loci: those that 1) depend solely on maintenance methylation machinery (NR loci), 2) depend on both maintenance methylation and RNAi components (R loci), and 3) depend solely on RNAi components (unaffected in ddm1 mutants). Furthermore, they speculate that this mechanism may allow for the generation of epialleles with differences in transgenerational stability.

Although, the authors performed a thorough analysis of the 500 kb heterchomatic knob region of chromosome 4, their conclusions could have strengthened if they would have performed a more genome-wide bisulfite sequencing approach to test whether remethylation occurs on a large-scale in both euchromatic and heterochromatic regions. Furthermore, it would have been informative if they would have reported whether small RNA levels increase with each generation. This may have yielded insight into the mechanism behind the progressive nature of remethylation. Several outstanding questions remain. What features distinguish remethylatable vs. non-remethylatable sequences? Why does it take so many generations for remethylation to be re-established? Is there a benefit for the observed slow re-establishment vs. a more rapid one?

Many X-linked microRNAs escape meiotic sex chromosome inactivation

Rui Song, Seungil Ro, Jason D Michaels, Chanjae Park, John R McCarrey and Wei Yan

Nature Genetics 41 (4): 488-93, April 2009.
doi:10.1038/ng.338

This week’s succinct summary and analysis provided by Anonymous:

Meiotic sex chromosome inactivation (MSCI) is a process that silences unsynapsed chromosomes during meiosis, specifically the X and Y chromosomes in males.  While the reasons for this silencing are not understood, it has been postulated that MSCI prevents genomic instability.  Following up on previously published observations, Song et al. investigated whether X-linked miRNA genes escape silencing.   Carrying out a real-time PCR-based survey of X-linked miRNAs, the authors found that numerous miRNAs were upregulated transcriptionally during stages of spermatogenesis in which MSCI is expected to function, suggesting a major distinction between transcriptional regulation of miRNAs and mRNAs.

This report systematically investigates all X-linked miRNAs in various stages of spermatogenesis, checks their transcriptional regulation to verify that increased levels are not due simply to increased stability of miRNAs transcribed prior to MSCI, and follows up on a few miRNAs in more detail using in situ hybridization.  One weakness of the study, however, is that absolute miRNA expression levels were not determined. Therefore, although a miRNA may be upregulated, it is unclear whether this upregulation results in  (1) full or only a fractional escape from MSCI, and (2) a biologically active concentration of miRNA.   Nevertheless, this study provides an interesting starting point to investigate differential regulation during MSCI.

Splicing Factors Facilitate RNAi-Directed Silencing in Fission Yeast

Elizabeth H. Bayne, Manuela Portoso, Alexander Kagansky, Isabelle C. Kos-Braun, Takeshi Urano, Karl Ekwall, Flavia Alves, Juri Rappsilber, Robin C. Allshire

Science 322 (5901): 602-606, October 2008.
doi: 10.1126/science.1164029

This week’s paper dissection by David Weinberg:

Centromeres in fission yeast (Schizosaccharomyces pombe) consist of a central kinetochore domain flanked by heterochromatic outer repeats. These outer repeats are transcribed by RNA polymerase II and maintained in a heterochromatic state by the RNAi pathway. According to the current model of S. pombe RNAi, the RDRC complex (Rdp1, Hrr1, and Cid12) converts nascent outer repeat transcripts into dsRNA, which serves as a substrate for Dcr1. siRNA-loaded Ago1, in complex with Tas3 and Chp1, is directed to complementary nascent transcripts and stimulates H3K9-dimethylation of histones by the Clr4 histone methyltranferase.

A previous forward genetic screen for ts lethal defects in centromere silencing had identified two components of the spliceosome, Cwf10 and Prp39. Based on this evidence, the authors hypothesized that there may be a link between splicing and centromere silencing in S. pombe. In this paper, they take a reverse genetics approach to further investigate this potential connection.

The paper begins by characterizing the extent of centromere silencing in a collection of ts lethal splicing mutants. They find that only specific splicing mutants show a defect in centromere silencing, i.e. there are many splicing mutants that show no effect. Among their mutant collection, they find a strong correlation between silencing of a centromere-embedded reporter gene, transcript levels of endogenous outer repeats, and quantities of outer repeat-derived siRNAs.

At this point, the authors point out that there are many possible “mundane” explanations for the effects they see, namely that splicing itself may affect mRNAs encoding proteins that are directly/indirectly involved in RNAi (including potentially Ago1 itself). Their numerous attempts to disprove this potential artifact are commendable – thorough, clever, cautious. While their data alone cannot entirely rule out the mundane, they did as much as could be done to convince the reader (and reviewers, presumably) that there must be interesting (i.e. splicing-independent) science at work here.

Figure 3 contains, by far, the least novel/interesting experiments of the paper. Basically the authors demonstrate that the centromere silencing defect in splicing mutants is due to a minor disruption in the maintenance of RNAi-dependent heterochromatin. The effect they see is surprisingly weak in comparison to a dcr1-null strain, suggesting that the role of the spliceosome is not essential for RNAi-directed heterochromatin.

Luckily, the paper ends on a high note with Figure 4. They use mass spec to show that Cid12 interacts with the RDRC complex, and chromatin immunoprecipitation to demonstrate an association between the spliceosome and centromere repeat DNA. In this way, the authors convert a nebulous genetic interaction between splicing and RNAi into a physical interaction between the spliceosome and the RNAi machinery. This interaction is also consistent with Figure 4B, which suggest that splicing factors act downstream of RITS recruitment (e.g. at the level of RDRC-dependent siRNA amplification).

This final set of experiments hint at mechanism and lead the authors to speculate that the spliceosome may provide a platform that facilitates RDRC recruitment/action. While this model is completely consistent with the data, there is little direct support for this model over a variety of other consistent models (which they entirely ignore). Along these lines, I was most intrigued by their initial observation that only specific splicing mutants showed the centromere silencing defect…but there was not even a mention of how this relates to their model. Still, the novel aspects of this paper – namely, the link between the spliceosome, but not splicing, and RNAi in the form of a physical interaction between the spliceosome and RDRC – make this an important paper in the field. Given the absence of RdRP machinery in metazoans, it will be interesting to see if a similar splicing-RNAi interaction is at work in higher eukaryotes.

nhl-2 Modulates MicroRNA Activity in Caenorhabditis elegans

Christopher M. Hammell, Isabella Lubin, Peter R. Boag, T. Keith Blackwell, and Victor Ambros

Cell 136 (5): 926–938, March 2009.
doi:10.1016/j.cell.2009.01.053

This week’s careful summary and analysis by Joel Neilson:

Lin-41 is a founding member of the heterochronic pathway and a member of the TRIM-NHL family of proteins.  The phenotype of the lin-41 loss- of-function is precocious development, but is not fully penetrant.  The authors were examining whether this phenotype could be accentuated by crossing in additional mutant alleles for other TRIM-NHL family  members.  In contrast to the accentuated phenotype they were expecting, they found that one of these loss-of-function mutants,  nhl-2, resulted in a mildly retarded heterochronic phenotype and rescued the defects in the lin-41 mutant.

To briefly summarize this study in the wrong order and a completely oversimplified manner, they then demonstrate that:

(1)  loss of nhl-2 gene function enhances the phenotype of individually non-penetrant LOF alleles for miRNAs in the let-7 family

(2)  loss of nhl-2 gene function enhances the phenotype of a weak LOF allele of a miRNA in a second family (lsy-6)

(3)  loss of nhl-2 gene function offsets phenotypes observed in animals ectopically expressing a let-7 family member

(4)  loss of nhl-2 gene function accentuates heterochronic defects in worms with mutations in core miRNA machinery components

(5)  all of this happens without modulation of the levels of mature miRNAs and is through previously characterized miRNA targets

(6)  NHL2 exhibits broad temporal-spatial expression

(7)  NHL2 co-localizes with and in fact touches CGH1.  They also genetically interact.

(8)  NHL-2 and CGH-1 physically interact with the core miRNA machinery in an
RNA-dependent fashion

(9)  CGH-1 still interacts with the core miRNA machinery in nhl-2 mutants.

This is a one of the best papers I have chosen for this forum and I got particularly excited upon reading the following in the introduction: “Current models do not adequately account for the facts that some miRNA targets appear to be regulated primarily at a translational level while others are regulated by mRNA turnover, or that a particular miRNA can have dramatically different potencies on distinct miRNA target reporters (Eulalio et al., 2007) therefore, it is likely that additional proteins can interact with miRISC to modulate the nature and efficacy of miRISC activity.”  Looking at the last clause of that sentence, they did in fact demonstrate that additional factors can modulate miRISC activity.  But that’s not what I got excited about in reading the introduction. To really nail down  the parts that current models do not adequately account for, someone really does need to show that a defined target, which sometimes (in a spatial or temporal manner) is affected one way by miRNA/RISC recognition. . .say, translational repression. . . and sometimes is  affected another way. . .say, deadenylation. . .by the same miRNA/RISC, and show what dictates this specificity.  This study did not directly address this issue but is definitely moving us in the right direction.