Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage.

To explore the role of Dicer-dependent control mechanisms in B lymphocyte development, we ablated this enzyme in early B cell progenitors. This resulted in a developmental block at the pro- to pre-B cell transition. Gene-expression profiling revealed a miR-17 approximately 92 signature in the 3'UTRs of genes upregulated in Dicer-deficient pro-B cells; a top miR-17 approximately 92 target, the proapoptotic molecule Bim, was highly upregulated. Accordingly, B cell development could be partially rescued by ablation of Bim or transgenic expression of the prosurvival protein Bcl-2. This allowed us to assess the impact of Dicer deficiency on the V(D)J recombination program in developing B cells. We found intact Ig gene rearrangements in immunoglobulin heavy (IgH) and kappa chain loci, but increased sterile transcription and usage of D(H) elements of the DSP family in IgH, and increased N sequence addition in Igkappa due to deregulated transcription of the terminal deoxynucleotidyl transferase gene.

Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions.

We use Caenorhabditis elegans to test proposed general rules for microRNA (miRNA)-target interactions. We show that G.U base pairing is tolerated in the 'seed' region of the lsy-6 miRNA interaction with its in vivo target cog-1, and that 6- to 8-base-pair perfect seed pairing is not a generally reliable predictor for an interaction of lsy-6 with a 3' untranslated region (UTR). Rather, lsy-6 can functionally interact with its target site only in specific 3' UTR contexts. Our findings illustrate the difficulty of establishing generalizable rules of miRNA-target interactions.

Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3'-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5'-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.

A physical and functional link between splicing factors promotes pre-mRNA 3' end processing.

Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3' end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3' end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation. We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins.

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3'UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.

A highly conserved cis-regulatory motif directs differential gonadal synexpression of Dmrt1 transcripts during gonad development.

Differential gene expression largely accounts for the coordinated manifestation of the genetic programme underlying embryonic development and cell differentiation. The 3' untranslated region (3'-UTR) of eukaryotic genes can contain motifs involved in regulation of gene expression at the post-transcriptional level. In the 3'-UTR of dmrt1, a key gene that functions in gonad development and differentiation, an 11-bp protein-binding motif was identified that mediates gonad-specific mRNA localization during embryonic and larval development of fish. Mutations that disrupt the 11-bp motif leading to in vitro protein-binding loss and selective transcript stabilization failure indicate a role for this motif in RNA stabilization through protein binding. The sequence motif was found to be conserved in most of the dmrt1 homologous genes from flies to humans suggesting a widespread conservation of this specific mechanism.

Allegro: analyzing expression and sequence in concert to discover regulatory programs.

A major goal of system biology is the characterization of transcription factors and microRNAs (miRNAs) and the transcriptional programs they regulate. We present Allegro, a method for de-novo discovery of cis-regulatory transcriptional programs through joint analysis of genome-wide expression data and promoter or 3' UTR sequences. The algorithm uses a novel log-likelihood-based, non-parametric model to describe the expression pattern shared by a group of co-regulated genes. We show that Allegro is more accurate and sensitive than existing techniques, and can simultaneously analyze multiple expression datasets with more than 100 conditions. We apply Allegro on datasets from several species and report on the transcriptional modules it uncovers. Our analysis reveals a novel motif over-represented in the promoters of genes highly expressed in murine oocytes, and several new motifs related to fly development. Finally, using stem-cell expression profiles, we identify three miRNA families with pivotal roles in human embryogenesis.

Identification of a signature motif in target mRNAs of RNA-binding protein AUF1.

The ubiquitous RNA-binding protein AUF1 promotes the degradation of some target mRNAs, but increases the stability and translation of other targets. Here, we isolated AUF1-associated mRNAs by immunoprecipitation of (AUF1-RNA) ribonucleoprotein (RNP) complexes from HeLa cells, identified them using microarrays, and used them to elucidate a signature motif shared among AUF1 target transcripts. The predicted AUF1 motif (29-39 nucleotides) contained 79% As and Us, consistent with the AU-rich sequences of reported AUF1 targets. Importantly, 10 out of 15 previously reported AUF1 target mRNAs contained the AUF1 motif. The predicted interactions between AUF1 and target mRNAs were recapitulated in vitro using biotinylated RNAs. Interestingly, further validation of predicted AUF1 target transcripts revealed that AUF1 associates with both the pre-mRNA and the mature mRNA forms. The consequences of AUF1 binding to 10 predicted target mRNAs were tested by silencing AUF1, which elevated the steady-state levels of only four mRNAs, and by overexpressing AUF1, which also lowered the levels of only four mRNAs. In total, we have identified a signature motif in AUF1 target mRNAs, have found that AUF1 also associates with the corresponding pre-mRNAs, and have discovered that altering AUF1 levels alone only modifies the levels of subsets of target mRNAs.

Mouse period 2 mRNA circadian oscillation is modulated by PTB-mediated rhythmic mRNA degradation.

Circadian mRNA oscillations are the main feature of core clock genes. Among them, period 2 is a key component in negative-feedback regulation, showing robust diurnal oscillations. Moreover, period 2 has been found to have a physiological role in the cell cycle or the tumor suppression. The present study reports that 3'-untranslated region (UTR)-dependent mRNA decay is involved in the regulation of circadian oscillation of period 2 mRNA. Within the mper2 3'UTR, both the CU-rich region and polypyrimidine tract-binding protein (PTB) are more responsible for mRNA stability and degradation kinetics than are other factors. Depletion of PTB with RNAi results in mper2 mRNA stabilization. During the circadian oscillations of mper2, cytoplasmic PTB showed a reciprocal expression profile compared with mper2 mRNA and its peak amplitude was increased when PTB was depleted. This report on the regulation of mper2 proposes that post-transcriptional mRNA decay mediated by PTB is a fine-tuned regulatory mechanism that includes dampening-down effects during circadian mRNA oscillations.

Long 3'-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway, present in most eukaryotic cells, is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons and, importantly, some wild-type mRNAs. Earlier studies demonstrated that aberrant mRNAs with artificially extended 3'-untranslated regions (3'-UTRs) are degraded by NMD. However, the extent to which wild-type mRNAs with long 3'-UTRs are degraded by NMD is not known. We used a global approach to identify wild-type mRNAs in Saccharomyces cerevisiae that have longer than expected 3'-UTRs, and of these mRNAs tested, 91% were degraded by NMD. We demonstrate for the first time that replacement of the natural, long 3'-UTR from wild-type PGA1 mRNA, which encodes a protein that is important for cell wall biosynthesis, with a short 3'-UTR renders it immune to NMD. The natural PGA1 3'-UTR is sufficient to target a NMD insensitive mRNA for decay by the NMD pathway. Finally, we show that nmd mutants are sensitive to Calcofluor White, which suggests that the regulation of PGA1 and other cell wall biosynthesis proteins by NMD is physiologically significant.