Calvin H. Jan

The Widespread Impact of Mammalian MicroRNAs on mRNA Repression and Evolution

Thousands of mammalian messenger RNAs are under selective pressure to maintain 7-nucleotide sites matching microRNAs (miRNAs). We found that these conserved targets are often highly expressed at developmental stages before miRNA expression and that their levels tend to fall as the miRNA that targets them begins to accumulate. Nonconserved sites, which outnumber the conserved sites 10 to 1, also mediate repression. As a consequence, genes preferentially expressed at the same time and place as a miRNA have evolved to selectively avoid sites matching the miRNA. This phenomenon of selective avoidance extends to thousands of genes and enables spatial and temporal specificities of miRNAs to be revealed by finding tissues and developmental stages in which messages with corresponding sites are expressed at lower levels.

Intronic microRNA precursors that bypass Drosha processing

MicroRNAs (miRNAs) are ∼22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs. In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a ∼60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. From that duplex, the mature miRNA is incorporated into the silencing complex. Here we identify an alternative pathway for miRNA biogenesis, in which certain debranched introns mimic the structural features of pre-miRNAs to enter the miRNA-processing pathway without Drosha-mediated cleavage. We call these pre-miRNAs/introns ‘mirtrons’, and have identified 14 mirtrons in Drosophila melanogaster and another four in Caenorhabditis elegans (including the reclassification of mir-62). Some of these have been selectively maintained during evolution with patterns of sequence conservation suggesting important regulatory functions in the animal. The abundance of introns comparable in size to pre-miRNAs appears to have created a context favourable for the emergence of mirtrons in flies and nematodes. This suggests that other lineages with many similarly sized introns probably also have mirtrons, and that the mirtron pathway could have provided an early avenue for the emergence of miRNAs before the advent of Drosha.

Conserved Function of lincRNAs in Vertebrate Embryonic Development despite Rapid Sequence Evolution

Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation.Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation.

Formation, Regulation and Evolution of Caenorhabditis elegans 3'UTRs

Post-transcriptional gene regulation frequently occurs through elements in mRNA 3′ untranslated regions (UTRs). Although crucial roles for 3′UTR-mediated gene regulation have been found in Caenorhabditis elegans, most C. elegans genes have lacked annotated 3′UTRs. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3′UTRs. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3′UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3′UTRs have median length only one-sixth that of mammalian 3′UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation.

A single Hox locus in Drosophila produces functional microRNAs from opposite DNA strands

MicroRNAs (miRNAs) are approximately 22-nucleotide RNAs that are processed from characteristic precursor hairpins and pair to sites in messages of protein-coding genes to direct post-transcriptional repression. Here, we report that the miRNA iab-4 locus in the Drosophila Hox cluster is transcribed convergently from both DNA strands, giving rise to two distinct functional miRNAs. Both sense and antisense miRNA products target neighboring Hox genes via highly conserved sites, leading to homeotic transformations when ectopically expressed. We also report sense/antisense miRNAs in mouse and find antisense transcripts close to many miRNAs in both flies and mammals, suggesting that additional sense/antisense pairs exist.

Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

The wheres and whys of protein translation Localized protein synthesis is important for a broad range of biological activities, from specifying the animal body plan to coordinating entry into the secretory pathway. Few tools are available that can investigate translation at specific subcellular sites. Jan et al. present a flexible ribosome profiling–based methodology to enable precise characterization of localized protein synthesis (see the Perspective by Shao and Hegde). Proximity-specific ribosome profiling provides a high-precision tool for looking at the mechanism of localized protein targeting and synthesis in living cells. The approach yielded a high-resolution systems-level view of cotranslational translocation at the endoplasmic reticulum. Williams et al. applied the technique to look at localized mRNA translation at the mitochondrial outer membrane. Science, this issue 10.1126/science.1257521, p. 701; see also p. 748 A new method reveals exactly which proteins are synthesized in the neighborhood of the endoplasmic reticulum and mitochondria. [Also see Perspective by Shao and Hegde] Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner-membrane proteins were cotranslationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein-folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.

Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling

Introduction Localized protein synthesis plays a critical role in creating subcellular structures by allowing protein production at the site of action and in response to local cellular need. Local translation is involved in diverse processes, including developmental patterning, cellular motility, synaptic plasticity, and protein trafficking through the secretory pathway. Despite this broad importance, few gene expression tools are available that faithfully preserve spatial information. We developed a flexible deep sequencing–based methodology (termed proximity-specific ribosome profiling) that enables precise characterization of localized protein synthesis. We applied our method to analyze translation at the endoplasmic reticulum (ER) in yeast and mammalian cells. Proximity-specific ribosome profiling provides spatiotemporal details of translation at the ER. Biotin ligase is localized to the ER as a fusion protein, where it biotinylates Avi-tagged ribosomes at the ER surface. Ribosome profiling is performed on streptavidinpurified ribosomes and compared to whole-cell profiling to resolve which genes are translated at the ER (1) and how much nascent chain was required to target the ribosome to the translocon (2).. Rationale The basis of our approach is to biotinylate ribosomes in intact cells in a manner dependent on their subcellular location. This is accomplished through the coexpression of a spatially restricted biotin ligase (BirA) fusion protein together with ribosomes containing an AviTag, which makes them substrates for BirA. Controlled pulses of biotin are then provided to allow for spatiotemporal control of ribosome labeling. This in vivo biotinylation enables the recovery of ribosomes from defined locations, including those that cannot be purified by classical cell fractionation techniques. Combining this purification strategy with ribosome profiling, the deep sequencing of ribosome-protected mRNA fragments, provides subcodon resolution of which messages were translated at the site of interest. Results We identified several principles used by cells to coordinate translation with ER targeting. Cotranslational targeting to the ER is pervasive and is principally determined by the location of the hydrophobic targeting sequence within the protein, rather than the mechanism of targeting or translocation. The position of this hydrophobic domain within the open reading frame determines the duration of time a targeted ribosome nascent-chain complex (RNC) can associate with the ER. Our data suggest a role for polysomes in retaining mRNAs at the ER, allowing for efficient targeting of RNCs for translocation. Position-specific analyses revealed that distinct translocon complexes engage nascent chains at different points during synthesis. Most proteins engage the ER immediately after or even before the signal sequence or signal anchor emerges from the ribosome. These nascent chains typically undergo a conformational rearrangement within the translocon, the proteinaceous tunnel through which nascent proteins cross the ER membrane. This rearrangement results in a “looped” conformation of the nascent chains, with their N termini facing the cytosol. This conformation is required for signal sequence processing. However, we discovered a class of Sec66-dependent proteins that engage only when they are long enough to adopt the looped conformation. Finally, we monitored the fate of ER-associated ribosomes after translation termination using pulsed-labeling experiments. These data demonstrated that ER-associated ribosomes readily exchanged into the cytosol after at most a few rounds of translation at the ER. Conclusion These results, together with those in an accompanying Report on translation at mitochondria, establish proximity-specific ribosome profiling as a robust and general tool. In principle, this method can be applied to any site that can be specified by a biotinligase fusion protein. Thus, our approach provides in vivo access to a broad spectrum of subpopulations of ribosomes defined either by their subcellular locations or through their interactions with specific factors, such as chaperones.. The wheres and whys of protein translation Localized protein synthesis is important for a broad range of biological activities, from specifying the animal body plan to coordinating entry into the secretory pathway. Few tools are available that can investigate translation at specific subcellular sites. Jan et al. present a flexible ribosome profiling–based methodology to enable precise characterization of localized protein synthesis (see the Perspective by Shao and Hegde). Proximity-specific ribosome profiling provides a high-precision tool for looking at the mechanism of localized protein targeting and synthesis in living cells. The approach yielded a high-resolution systems-level view of cotranslational translocation at the endoplasmic reticulum. Williams et al. applied the technique to look at localized mRNA translation at the mitochondrial outer membrane. Science, this issue 10.1126/science.1257521, p. 701; see also p. 748 A new method reveals exactly which proteins are synthesized in the neighborhood of the endoplasmic reticulum and mitochondria. [Also see Perspective by Shao and Hegde] Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of posttranslational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

Response to Comment on “Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling”

Reid and Nicchitta propose that most cellular translation is carried out by a noncycling pool of endoplasmic reticulum (ER)–associated ribosomes. However, proximity-specific ribosome profiling data place an upper bound of about 7 to 16% on the fraction of cytosolic protein translation carried out by ribosomes accessible to ER-tethered biotin ligases. Moreover, yeast pulse-labeling experiments argue against there being a static population of ER-associated ribosomes.

Lipid Homeostasis Is Maintained by Dual Targeting of the Mitochondrial PE Biosynthesis Enzyme to the ER

Spatial organization of phospholipid synthesis in eukaryotes is critical for cellular homeostasis. The synthesis of phosphatidylcholine (PC), the most abundant cellular phospholipid, occurs redundantly via the ER-localized Kennedy pathway and a pathway that traverses the ER and mitochondria via membrane contact sites. The basis of the ER-mitochondrial PC synthesis pathway is the exclusive mitochondrial localization of a key pathway enzyme, phosphatidylserine decarboxylase Psd1, which generates phosphatidylethanolamine (PE). We find that Psd1 is localized to both mitochondria and the ER. Our data indicate that Psd1-dependent PE made at mitochondria and the ER has separable cellular functions. In addition, the relative organellar localization of Psd1 is dynamically modulated based on metabolic needs. These data reveal a critical role for ER-localized Psd1 in cellular phospholipid homeostasis, question the significance of an ER-mitochondrial PC synthesis pathway to cellular phospholipid homeostasis, and establish the importance of fine spatial regulation of lipid biosynthesis for cellular functions.