Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo.

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

Techniques for following the movement of single RNAs in living cells.

The ability to investigate gene expression has evolved from static approaches that analyze a population of cells to dynamic approaches that analyze individual living cells. During the last decade, a number of different fluorescent methods have been developed for monitoring the dynamics of single RNAs in living cells. Spatial-temporal analyses of single RNAs in living cells have provided novel insight into nuclear transport, RNA localization, and decay. Technical advances with these approaches allow for single molecule detection, providing an unprecedented view of RNA movement. In this article, we discuss the methods for observing single RNAs in living cells, highlighting the advantages and limitations of each method.

Monitoring the temporal and spatial distribution of RNA in living yeast cells.

RNA localization is a cellular process to spatially restrict translation of specific proteins to defined regions within or between cells. Most localized mRNAs contain cis-acting localization elements in the 3'-untranslated region (UTR), which are sufficient for localization of an mRNA to a particular region of the cell. The cis-acting localization elements serve as assembly sites for trans-acting factors which function to sort the mRNA to the correct sub-cellular destination. Although fluorescent in situ hybridization (FISH) has been widely used to study mRNA localization, FISH has a weakness in that it is a static assay, as FISH requires that cells be fixed before hybridization. Consequently, FISH is not ideally suited for investigating dynamic mRNA localization processes. This limitation of FISH has been overcome by the development of techniques that allow the visualization of mRNA in living cells. Here, we present a protocol that tethers green fluorescent protein (GFP) to an mRNA of interest, allowing for the visualization of dynamic mRNA localization processes in living cells.

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit.

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in "half-mer" polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Delta cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Delta cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Delta cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.

RNA localization in yeast: moving towards a mechanism.

RNA localization is a widely utilized strategy employed by cells to spatially restrict protein function. In Saccharomyces cerevisiae asymmetric sorting of mRNA to the bud has been reported for at least 24 mRNAs. The mechanism by which the mRNAs are trafficked to the bud, illustrated by ASH1 mRNA, involves recognition of cis-acting localization elements present in the mRNA by the RNA-binding protein, She2p. The She2p/mRNA complex subsequently associates with the myosin motor protein, Myo4p, through an adapter, She3p. This ribonucleoprotein complex is transported to the distal tip of the bud along polarized actin cables. While the mechanism by which ASH1 mRNA is anchored at the bud tip is unknown, current data point to a role for translation in this process, and the rate of translation of Ash1p during the transport phase is regulated by the cis-acting localization elements. Subcellular sorting of mRNA in yeast is not limited to the bud; certain mRNAs corresponding to nuclear-encoded mitochondrial proteins are specifically sorted to the proximity of mitochondria. Analogous to ASH1 mRNA localization, mitochondrial sorting requires cis-acting elements present in the mRNA, though trans-acting factors involved with this process remain to be identified. This review aims to discuss mechanistic details of mRNA localization in S. cerevisiae.

Identification of NH...N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy.

RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH...N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6-2.9 A. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH.N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.

Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3.

The RNA-mediated pathogenesis model for the myotonic dystrophies DM1 and DM2 proposes that mutant transcripts from the affected genes sequester a family of double-stranded RNA-binding factors, the muscleblind proteins MBNL1, MBNL2 and MBNL3, in the nucleus. These proteins are homologues of the Drosophila muscleblind proteins that are required for the terminal differentiation of muscle and photoreceptor tissues, and thus nuclear sequestration of the human proteins might impair their normal function in muscle and eye development and maintenance. To examine this model further, we analyzed the expression pattern of the mouse Mbnl1, Mbnl2, and Mbnl3 genes during embryonic development and compared muscleblind gene expression to Dmpk since the RNA pathogenesis model for DM1 requires the coordinate synthesis of mutant Dmpk transcripts and muscleblind proteins. Our studies reveal a striking overlap between the expression of Dmpk and the muscleblind genes during development of the limbs, nervous system and various muscles, including the diaphragm and tongue.

Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export.

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3'-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)(+) RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Delta hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export.