Howard D. Lipshitz

A compendium of RNA-binding motifs for decoding gene regulation

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

The maternal-to-zygotic transition: a play in two acts

All animal embryos pass through a stage during which developmental control is handed from maternally provided gene products to those synthesized from the zygotic genome. This maternal-to-zygotic transition (MZT) has been extensively studied in model organisms, including echinoderms, nematodes, insects, fish, amphibians and mammals. In all cases, the MZT can be subdivided into two interrelated processes: first, a subset of maternal mRNAs and proteins is eliminated; second, zygotic transcription is initiated. The timing and scale of these two events differ across species, as do the cellular and morphogenetic processes that sculpt their embryos. In this article, we discuss conserved and distinct features within the two component processes of the MZT.

Vectors for Drosophila P-element-mediated transformation and tissue culture transfection

We describe nine P-element vectors that can be used to study gene regulation and function in Drosophila. These vectors were designed for use in germline transformation and cell culture transfection assays. One set consists of five P elements that can be used to study transcriptional regulatory sequences. These vectors contain several unique restriction sites for insertion of a foreign promoter upstream from either a cat or lacZ reporter gene. Two of the beta-galactosidase-coding vectors also require the insertion of a start codon for translation of the reporter enzyme and thus can be used to study translational regulatory sequences. The second set of P elements consists of four vectors that contain the Drosophila cytoplasmic actin 5C promoter and polyadenylation signals. Upon insertion of a foreign DNA segment, these vectors direct constitutive expression of the encoded RNA and protein.

A Vertebrate Polycomb Response Element Governs Segmentation of the Posterior Hindbrain

Chromatin remodeling by Polycomb group (PcG) and trithorax group (trxG) proteins regulates gene expression in all metazoans. Two major complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), are thought to mediate PcG-dependent repression in flies and mammals. In Drosophila, PcG/trxG protein complexes are recruited by PcG/trxG response elements (PREs). However, it has been unclear how PcG/trxG are recruited in vertebrates. Here we have identified a vertebrate PRE, PRE-kr, that regulates expression of the mouse MafB/Kreisler gene. PRE-kr recruits PcG proteins in flies and mouse F9 cells and represses gene expression in a PcG/trxG-dependent manner. PRC1 and 2 bind to a minimal PRE-kr region, which can recruit stable PRC1 binding but only weak PRC2 binding when introduced ectopically, suggesting that PRC1 and 2 have different binding requirements. Thus, we provide evidence that similar to invertebrates, PREs act as entry sites for PcG/trxG chromatin remodeling in vertebrates.

Joint action of two RNA degradation pathways controls the timing of maternal transcript elimination at the midblastula transition in Drosophila melanogaster

Maternally synthesized RNAs program early embryonic development in many animals. These RNAs are degraded rapidly by the midblastula transition (MBT), allowing genetic control of development to pass to zygotically synthesized transcripts. Here we show that in the early embryo of Drosophila melanogaster, there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is targeted to specific classes of mRNAs through cis‐acting elements in the 3′‐untranslated region and is conserved in Xenopus laevis. The second pathway is activated 2 h after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT.

Mechanisms of RNA localization and translational regulation.

Transcript localization and translational regulation are two post-transcriptional mechanisms for the spatial and temporal regulation of protein production. During the past year, two transcript localization mechanisms have been elaborated in some detail. Where localization involves directional transport on cytoskeletal tracks, links between the transcripts and the cytoskeletal molecular motors have been elaborated. In the case of localization by generalized transcript degradation combined with localized protection, trans-acting pathways and cis-acting elements for degradation and protection have been identified. A third transcript localization mechanism, vectorial transport out of the nucleus into a particular cytoplasmic domain, was initially thought to localize pair-rule transcripts in Drosophila. However, these have now been shown to be localized by directional transport in the cytoplasm. Transcript localization and translational regulation can be intimately linked in that, for certain messenger RNAs, only the localized fraction of transcripts is translated whereas unlocalized transcripts are translationally repressed. Cis-acting sequences and trans-acting factors that function in translational repression have been identified along with factors involved in relief of translational repression at the site of localization.

Spatial and temporal control of RNA stability.

Maternally encoded RNAs and proteins program the early development of all animals. A subset of the maternal transcripts is eliminated from the embryo before the midblastula transition. In certain cases, transcripts are protected from degradation in a subregion of the embryonic cytoplasm, thus resulting in transcript localization. Maternal factors are sufficient for both the degradation and protection components of transcript localization. Cis-acting elements in the RNAs convert transcripts progressively (i) from inherently stable to unstable and (ii) from uniformly degraded to locally protected. Similar mechanisms are likely to act later in development to restrict certain classes of transcripts to particular cell types within somatic cell lineages. Functions of transcript degradation and protection are discussed.

Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate

BACKGROUND Drosophila numb was originally described as a mutation affecting binary divisions in the sensory organ precursor (SOP) lineage. The numb gene was subsequently shown to encode an asymmetrically localized protein which is required for binary cell-fate decisions during peripheral nervous system development. Part of the Drosophila NUMB protein exhibits homology to the SHC phosphotyrosine-binding (PTB) domain, suggesting a potential link to tyrosine-kinase signal transduction. RESULTS A widely expressed mammalian homologue of Drosophila numb (dnumb) has been cloned from rat and is referred to here as mammalian Numb (mNumb). The mNUMB protein has a similar overall structure to dNUMB and 67 sequence similarity. Misexpression of mNumb in Drosophila during sensory nervous system precursor cell division causes identical cell fate transformations to those produced by ectopic dNUMB expression. In vitro, the mNUMB PTB domain binds phosphotyrosine-containing proteins, and SH3 domains of SRC-family tyrosine kinases bind to mNUMB presumably through interactions with proline-rich regions in the carboxyl terminus. Overexpression of full-length mNUMB in the multipotential neural crest stem cell line MONC-1 dramatically biases its differentiation towards neurons, whereas overexpression of the mNUMB PTB domain biases its differentiation away from neuronal fates. CONCLUSIONS Our results demonstrate that mNUMB is an evolutionarily conserved functional homologue of dNUMB, and establish a link to tyrosine-kinase-mediated signal transduction pathways. Furthermore, our results suggest that mNUMB and dNUMB are new members of a family of signaling adapter molecules that mediate conserved cell-fate decisions during development.

Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus‐silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV‐1) vectors dominantly silence a linked locus control region (LCR) β‐globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus‐transduced embryonic stem (ES) cells are silenced predominantly 3 days post‐infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase‐null [dnmt3a−/−;dnmt3b−/−] ES cells. MSCV and HIV‐1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.

Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis

Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution.