Gordon G. Carmichael

The Fate of dsRNA in the Nucleus: A p54nrb-Containing Complex Mediates the Nuclear Retention of Promiscuously A-to-I Edited RNAs

How do cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously edited RNAs that encode nonfunctional, mutant proteins? We have developed a Xenopus oocyte model system which shows that a variety of hyperedited, inosine-containing RNAs are specifically retained in the nucleus. To uncover the mechanism of inosine-induced retention, HeLa cell nuclear extracts were used to isolate a multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54(nrb), the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. We provide evidence that one function of the complex identified here is to anchor hyperedited RNAs to the nuclear matrix, while allowing selectively edited mRNAs to be exported.

An alternative pathway for gene regulation by Myc

The c‐Myc protein activates transcription as part of a heteromeric complex with Max. However, Myc‐transformed cells are characterized by loss of expression of several genes, suggesting that Myc may also repress gene expression. Two‐hybrid cloning identifies a novel POZ domain Zn finger protein (Miz‐1; Myc‐interacting Zn finger protein‐1) that specifically interacts with Myc, but not with Max or USF. Miz‐1 binds to start sites of the adenovirus major late and cyclin D1 promoters and activates transcription from both promoters. Miz‐1 has a potent growth arrest function. Binding of Myc to Miz‐1 requires the helix–loop–helix domain of Myc and a short amphipathic helix located in the carboxy‐terminus of Miz‐1. Expression of Myc inhibits transactivation, overcomes Miz‐1‐induced growth arrest and renders Miz‐1 insoluble in vivo. These processes depend on Myc and Miz‐1 association and on the integrity of the POZ domain of Miz‐1, suggesting that Myc binding activates a latent inhibitory function of this domain. Fusion of a nuclear localization signal induces efficient nuclear transport of Miz‐1 and impairs the ability of Myc to overcome transcriptional activation and growth arrest by Miz‐1. Our data suggest a model for how gene repression by Myc may occur in vivo.

Quality Control of mRNA Function

While individual nuclear and cytoplasmic reactions required for the formation of functional mRNA can be carried out in isolation in vitro, it has become increasingly clear that many of the steps along the path from gene to protein are, in vivo, interdependent in a way that provides important mechanisms for the QC of mRNA function. In fact, nothing less would be expected of an efficiently operating assembly line, which should discard defective products rather than proceed to process them. Owing to space and reference constraints, this minireview describes neither the bulk of data demonstrating the interdependence of reactions required for mRNA biosynthesis, nor the finer details of these reactions. Future work will no doubt reveal the complete network of integrated events that ensures mRNA QC and yet-to-be-defined molecular constituents of this network.‡E-mail: lynne_maquat@urmc.rochester.edu (L. E. M.); gcarmich@neuron.uchc.edu (G. G. C.).

Alu element-mediated gene silencing

The Alu elements are conserved ∼300‐nucleotide‐long repeat sequences that belong to the SINE family of retrotransposons found abundantly in primate genomes. Pairs of inverted Alu repeats in RNA can form duplex structures that lead to hyperediting by the ADAR enzymes, and at least 333 human genes contain such repeats in their 3′‐UTRs. Here, we show that a pair of inverted Alus placed within the 3′‐UTR of egfp reporter mRNA strongly represses EGFP expression, whereas a single Alu has little or no effect. Importantly, the observed silencing correlates with A‐to‐I RNA editing, nuclear retention of the mRNA and its association with the protein p54nrb. Further, we show that inverted Alu elements can act in a similar fashion in their natural chromosomal context to silence the adjoining gene. For example, the Nicolin 1 gene expresses multiple mRNA isoforms differing in the 3′‐UTR. One isoform that contains the inverted repeat is retained in the nucleus, whereas another lacking these sequences is exported to the cytoplasm. Taken together, these results support a novel role for Alu elements in human gene regulation.

Genome‐Wide Studies Reveal That Lin28 Enhances the Translation of Genes Important for Growth and Survival of Human Embryonic Stem Cells

Lin28 inhibits the expression of let‐7 microRNAs but also exhibits let‐7‐independent functions. Using immunoprecipitation and deep sequencing, we show here that Lin28 preferentially associates with a small subset of cellular mRNAs. Of particular interest are those for ribosomal proteins and metabolic enzymes, the expression levels of which are known to be coupled to cell growth and survival. Polysome profiling and reporter analyses suggest that Lin28 stimulates the translation of many or most of these targets. Moreover, Lin28‐responsive elements were found within the coding regions of all target genes tested. Finally, a mutant Lin28 that still binds RNA but fails to interact with RNA helicase A (RHA), acts as a dominant‐negative inhibitor of Lin28‐dependent stimulation of translation. We suggest that Lin28, working in concert with RHA, enhances the translation of genes important for the growth and survival of human embryonic stem cells. STEM CELLS 2011;496–504

Decoding the function of nuclear long non-coding RNAs

Long non-coding RNAs (lncRNAs) are mRNA-like, non-protein-coding RNAs that are pervasively transcribed throughout eukaryotic genomes. Rather than silently accumulating in the nucleus, many of these are now known or suspected to play important roles in nuclear architecture or in the regulation of gene expression. In this review, we highlight some recent progress in how lncRNAs regulate these important nuclear processes at the molecular level.

INTRONLESS MRNA TRANSPORT ELEMENTS MAY AFFECT MULTIPLE STEPS OF PRE-MRNA PROCESSING

We have reported recently that a small element within the mouse histone H2a‐coding region permits efficient cytoplasmic accumulation of intronless β‐globin cDNA transcripts. This sequence lowers the levels of spliced products from intron‐containing constructs and can functionally replace Rev and the Rev‐responsive element (RRE) in the nuclear export of unspliced HIV‐1‐related mRNAs. In work reported here, we further investigate the molecular mechanisms by which this element might work. We demonstrate here through both in vivo and in vitro assays that, in addition to promoting mRNA nuclear export, this element acts as a polyadenylation enhancer and as a potent inhibitor of splicing. Surprisingly, two other described intronless mRNA transport elements (from the herpes simplex virus thymidine kinase gene and hepatitis B virus) appear to function in a similar manner. These findings prompt us to suggest that a general feature of intronless mRNA transport elements might be a collection of phenotypes, including the inhibition of splicing and the enhancement of both polyadenylation and mRNA export.

Functional interaction between RAFT1/FRAP/mTOR and protein kinase Cδ in the regulation of cap-dependent initiation of translation

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E‐BP1). The rapamycin and FK506‐binding protein (FKBP)‐target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E‐BP1. However, the molecular mechanisms involved in growth factor‐initiated phosphorylation of 4E‐BP1 are not well understood. Here we demonstrate that protein kinase Cδ (PKCδ) associates with RAFT1 and that PKCδ is required for the phosphorylation and inactivation of 4E‐BP1. PKCδ‐mediated phosphorylation of 4E‐BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E‐BP1 by PKCδ inhibits the interaction between 4E‐BP1 and eIF4E and stimulates cap‐dependent translation. Moreover, a dominant‐negative mutant of PKCδ inhibits serum‐induced phosphorylation of 4E‐BP1. These findings demonstrate that PKCδ associates with RAFT1 and thereby regulates phosphorylation of 4E–BP1 and cap‐dependent initiation of protein translation.

Vigilins bind to promiscuously A-to-I-edited RNAs and are involved in the formation of heterochromatin

The fate of double-stranded RNA (dsRNA) in the cell depends on both its length and location . The expression of dsRNA in the nucleus leads to several distinct consequences. First, the promiscuous deamination of adenosines to inosines by dsRNA-specific adenosine deaminase (ADAR) can lead to the nuclear retention of edited transcripts . Second, dsRNAs might induce heterochromatic gene silencing through an RNAi-related mechanism . Is RNA editing also connected to heterochromatin? We report that members of the conserved Vigilin class of proteins have a high affinity for inosine-containing RNAs. In agreement with other work , we find that these proteins localize to heterochromatin and that mutation or depletion of the Drosophila Vigilin, DDP1, leads to altered nuclear morphology and defects in heterochromatin and chromosome segregation. Furthermore, nuclear Vigilin is found in complexes containing not only the editing enzyme ADAR1 but also RNA helicase A and Ku86/70. In the presence of RNA, the Vigilin complex recruits the DNA-PKcs enzyme, which appears to phosphorylate a discrete set of targets, some or all of which are known to participate in chromatin silencing. These results are consistent with a mechanistic link between components of the DNA-repair machinery and RNA-mediated gene silencing.

Effects of Length and Location on the Cellular Response to Double-Stranded RNA

SUMMARY Since double-stranded RNA (dsRNA) has not until recently generally been thought to be deliberately expressed in cells, it has commonly been assumed that the major source of cellular dsRNA is viral infections. In this view, the cellular responses to dsRNA would be natural and perhaps ancient antiviral responses. While the cell may certainly react to some dsRNAs as an antiviral response, this does not represent the only response or even, perhaps, the major one. A number of recent observations have pointed to the possibility that dsRNA molecules are not seen only as evidence of viral infection or recognized for degradation because they cannot be translated. In some instances they may also play important roles in normal cell growth and function. The purpose of this review is to outline our current understanding of the fate of dsRNA in cells, with a focus on the apparent fact that their fates and functions appear to depend critically not only on where in the cell dsRNA molecules are found, but also on how long they are and perhaps on how abundant they are.