Andrea Du Toit

Non-coding RNA: RNA stability control by Pol II

(Pol II) is a DNA-dependent RNA polymerase that synthesizes mRNAs and non-coding RNAs (ncRNAs). Although Pol II can also function as an RNA-dependent RNA polymerase (RdRP), natural cellular RNA substrates have not yet been identified. Wagner et al. now shed light on the cellular function of Pol II RdRP activity by identifying an ncRNA that is extended and thereby destabilized by Pol II. The mouse ncRNA B2 RNA is transcribed by Pol III, and its levels are increased in response to stress such as heat shock. B2 RNA then represses Pol II-transcribed genes by interacting with a region in Pol II that overlaps with its DNA-binding cleft, thus disrupting polymerase–DNA contacts. In the new study, Wagner et al. showed that the dissociation of B2 RNA from human Pol II by an activity in nuclear extracts requires all four NTPs and Pol II catalytic activity, which led the authors to propose that B2 RNA serves as a substrate for the RdRP activity of Pol II. Indeed, using biochemical studie s in the absence and presence of the Pol II inhibitor α-amanitin, the authors showed that Pol II can extend the native 3ʹ end of B2 RNA by 18 nucleotides. Moreover, this extension was found to be a single-round reaction and to involve an internal template sequence near the 3ʹ end of B2 RNA. Next, the authors showed that extended B2 RNA maintained the ability to repress DNA-dependent transcription by Pol II in both a reconstituted in vitro system and transiently transfected COS cells, although repression by extended B2 RNA was less potent than that by B2 RNA. Consistent with this, site-specific protein–DNA photocrosslinking assays revealed that extended B2 RNA inhibited the interaction of the Pol II subunit RPB1 with promoter DNA, albeit to a lesser extent than B2 RNA. To delineate the cellular role of B2 RNA extension by Pol II, the authors first confirmed the presence of extended B2 RNA in NIH 3T3 cells using reverse transcription PCR and then tested the effect of α-amanitin on B2 RNA levels. Unexpectedly, the authors found that total B2 RNA levels (but not the levels of other Pol III transcripts) were greatly increased, whereas extended B2 RNA levels did not change. The authors excluded the possibility that this was due to increased stress-induced Pol IIImediated transcription. Rather, they suggested that the extension of 3ʹ ends by the RdRP activity of Pol II destabilizes B2 RNA, as inhibition of this activity by α-amanitin resulted in an increase in the steady-state levels of B2 RNA. This was confirmed, as extended B2 RNA was much less stable than total B2 RNA in cells treated with the transcription inhibitor actinomycin D. Together, this study provides evidence that a mammalian ncRNA serves as a substrate for Pol II and demonstrates that the RdRP activity of Pol II can control RNA stability by extending its 3ʹ end. This raises the intriguing question of what physiological role this control of B2 RNA stability might have and whether Pol II can also target other cellular RNAs. Andrea Du Toit N O N C O D I N G R N A

Endocytosis: A new gateway into cells

Two studies have determined the molecular mechanisms that underlie a specific clathrin-independent pathway for the internalization of activated surface receptors and bacterial toxins.Two studies have determined the molecular mechanisms that underlie a specific clathrin-independent pathway for the internalization of activated surface receptors and bacterial toxins.

Mechanotransduction: VE-cadherin lets it flow

This study shows that the transmembrane domain of VE-cadherin binds to VEGFR2 and VEGFR3, a new component of the junction mechanosensory complex.

RNA interference: Nuclear Dicer makes the cut

Mammalian Dicer is recruited to loci of endogenously overlapping transcripts to promote the formation of heterochromatin.

Organelle dynamics: KIF7 organizes cilia

KIF7 binds to microtubule plus ends at cilia tips to control cilium architecture and to organize a specialized compartment that mediates Hedgehog signalling.

Endocytosis: Bend it like galectin 3

Galectin 3 binds to glycosylated cargos and glycosphingolipids, which leads to membrane bending and clathrin-independent carrier biogenesis.

Microscopy: Advancing imaging

Betzig and colleagues created ultrathin light sheets to visualize dynamic processes at high speed and high spatiotemporal resolution.

Cell death: Balance through a bivalent regulator

SPTF-3 promotes cell type-specific apoptosis through caspase-dependent and caspase-independent pathways.

A new Hippo pathway component: Cell signalling

MASK is a novel and conserved component of the Hippo pathway that is required for Yorkie activity.

RNA: Expanding the mRNA epitranscriptome

Two new studies report the identification and comprehensive analysis of the m1A mRNA modification, showing that this mark is enriched at the 5′ UTRs of mRNAs and might have a functional role in promoting translation of methylated transcripts.