Mapping domains of ARS2 critical for its RNA decay capacity.

ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5'cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice.

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

MicroRNA-148b regulates megalin expression and is associated with receptor downregulation in mice with unilateral ureteral obstruction.

Megalin is a multiligand, endocytic receptor that is important for the normal, proximal tubule reabsorption of filtered proteins, hormones, enzymes, essential nutrients, and nephrotoxins. Megalin dysfunction has been associated with acute, as well as chronic kidney diseases. Tubular proteinuria has been observed following unilateral ureteral obstruction (UUO), suggesting megalin dysfunction; however, the pathophysiological mechanism has not been determined. To identify potential regulators of megalin expression, we examined renal microRNAs (miRNAs) expression and observed an upregulation of microRNA-148b (miR-148b) in obstructed mouse kidneys 7 days after UUO, which was associated with a significant reduction in proximal tubule megalin expression and accumulation of megalin ligands. By in silico miRNA target prediction analysis, we identified megalin messenger RNA (mRNA) as a potential target of miR-148b and confirmed using a dual-luciferase reporter assay that miR-148b targeted the 3'-untranslated region of the megalin gene. Transfection of LLC-PK1 cells with miR-148b mimic reduced endogenous megalin mRNA and protein levels in a concentration-dependent manner, while transfection with miR-148b inhibitor resulted in an increase. Our findings suggest that miR-148b directly downregulates megalin expression and that miR-148b negatively regulates megalin expression in UUO-induced kidney injury. Furthermore, the identification of a miRNA regulating megalin expression may allow for targeted interventions to modulate megalin function and proximal tubule uptake of proteins, as well as other ligands.

Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality.

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3' ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Making ends meet: coordination between RNA 3'-end processing and transcription initiation.

RNA polymerase II (RNAPII)-mediated gene transcription initiates at promoters and ends at terminators. Transcription termination is intimately connected to 3'-end processing of the produced RNA and already when loaded at the promoter, RNAPII starts to become configured for this downstream event. Conversely, RNAPII is 'reset' as part of the 3'-end processing/termination event, thus preparing the enzyme for its next round of transcription--possibly on the same gene. There is both direct and circumstantial evidence for preferential recycling of RNAPII from the gene terminator back to its own promoter, which supposedly increases the efficiency of the transcription process under conditions where RNAPII levels are rate limiting. Here, we review differences and commonalities between initiation and 3'-end processing/termination processes on various types of RNAPII transcribed genes. In doing so, we discuss the requirements for efficient 3'-end processing/termination and how these may relate to proper recycling of RNAPII.

Promoter-proximal polyadenylation sites reduce transcription activity.

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ~500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.

A pause to splice.

Maturation of mRNA termini occurs during transcription and can be aided by pausing of RNA polymerase II (RNAPII). In this issue of Molecular Cell, two groups now demonstrate that RNAPII pausing may also assist cotranscriptional splicing in S. cerevisiae.