Summary Cleavage Factor Im (CFIm) is a highly conserved component of the eukaryotic mRNA 3′ processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of two protein subunits, a 25 kDa subunit containing a nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFIm25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here we report the crystal structure of CFIm25 complexed with the RRM domain of CFIm68 and RNA. The CFIm25 dimer is clasped on opposite sides by two CFIm68 RRM domains. Each CFIm25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFIm68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFIm to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.

Human Cleavage Factor Im (CFI m ) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI m 25) of the CFI m complex possesses a characteristic α/β/α Nudix fold, CFI m 25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI m 25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI m 25 is the first Nudix protein to be reported to bind RNA in a sequencespecific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap 4 A (diadenosine tetraphosphate) by CFI m 25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing.T he transcriptome complexity of higher eukaryotes requires the coordinate recognition of an array of alternative pre-mRNA processing signals in a developmental and tissue-specific manner (1, 2). The sequences that direct pre-mRNA splicing and 3′ processing are initially recognized within the nascent transcript in a process that is intimately coupled to transcription (3, 4). While the recognition of exons within the pre-mRNA is mediated by both RNA:RNA and protein:RNA interactions (5), the 3′ processing of polyadenylated mRNAs appears to rely solely on the interaction of protein factors (6) with unstructured RNA sequences (7) within the nascent transcript.Vertebrate pre-mRNA 3′ processing signals are recognized by a tripartite mechanism through which a set of short RNA sequences direct the cooperative binding of three multimeric 3′ processing factors, cleavage factor I m (CFI m ), cleavage and polyadenylation specificity factor (CPSF), and cleavage stimulation factor (CstF) (8). CPSF and CstF bind the AAUAAA hexamer and downstream GU-rich elements that flank the poly(A) site, respectively, whereas CFI m interacts with upstream sequences that may function in the regulation of alternative polyadenylation (9-11). SELEX and biochemical analyses have identified the sequence UGUAN (N ¼ A > U > G, C) as the preferred binding site of CFI m (11). In this report we have taken a structural approach to determine the mechanism of sequence-specific RNA binding by CFI m .CFI m is composed of a large subunit of 59, 68, or 72 kDa and a small subunit of 25 kDa (CFI m 25, also referred to as CPSF5 or NUDT21) (12, 13), both of which contribute to RNA binding (14). The large subunit, encoded by either of two paralogs (CPSF6 and CPSF7), contains an N-terminal RNA Recognition Motif (RRM), an internal polyproline-rich region, and a C-termina...

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1alpha cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1alpha in pancreatic beta cells. Expression of collectrin was decreased in the islets of HNF-1alpha (-/-) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the beta cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca(2+) influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.

One subtype of maturity-onset diabetes of the young (MODY)-3 results from mutations in the gene encoding hepatocyte nuclear factor (HNF)-1␣. We generated transgenic mice expressing a naturally occurring dominant-negative form of human HNF-1␣ (P291fsinsC) in pancreatic ␤-cells. A progressive hyperglycemia with age was seen in these transgenic mice, and the mice developed diabetes with impaired glucose-stimulated insulin secretion. The pancreatic islets exhibited abnormal architecture with reduced expression of glucose transporter (GLUT2) and E-cadherin. Blockade of Ecadherin-mediated cell adhesion in pancreatic islets abolished the glucose-stimulated increases in intracellular Ca 2؉ levels and insulin secretion, suggesting that loss of E-cadherin in ␤-cells is associated with impaired insulin secretion. There was also a reduction in ␤-cell number (50%), proliferation rate (15%), and pancreatic insulin content (45%) in 2-day-old transgenic mice and a further reduction in 4-week-old animals. Our findings suggest various roles for HNF-1␣ in normal glucose metabolism, including the regulation of glucose transport, ␤-cell growth, and ␤-cell-to-␤-cell communication.

Germ cells develop in a microenvironment created by the somatic cells of the gonad [1-3]. Although in males, the germ and somatic support cells lie in direct contact, in females, a thick extracellular coat surrounds the oocyte, physically separating it from the somatic follicle cells [4]. To bypass this barrier to communication, narrow cytoplasmic extensions of the follicle cells traverse the extracellular coat to reach the oocyte plasma membrane [5-9]. These delicate structures provide the sole platform for the contact-mediated communication between the oocyte and its follicular environment that is indispensable for production of a fertilizable egg [8, 10-15]. Identifying the mechanisms underlying their formation should uncover conserved regulators of fertility. We show here in mice that these structures, termed transzonal projections (TZPs), are specialized filopodia whose number amplifies enormously as oocytes grow, enabling increased germ-soma communication. By creating chimeric complexes of genetically tagged oocytes and follicle cells, we demonstrate that follicle cells elaborate new TZPs that push through the extracellular coat to reach the oocyte surface. We further show that growth-differentiation factor 9, produced by the oocyte, drives the formation of new TZPs, uncovering a key yet unanticipated role for the germ cell in building these essential bridges of communication. Moreover, TZP number and germline-soma communication are strikingly reduced in reproductively aged females. Thus, the growing oocyte locally remodels follicular architecture to ensure that its developmental needs are met, and an inability of somatic follicle cells to respond appropriately to oocyte-derived cues may contribute to human infertility.

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The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, lifethreatening conditions. In response, here we present an electrochemical aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, we have developed an electrochemical, aptamer-based (EAB) sensor against Neutrophil Gelatinase-Associated Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clinically relevant NGAL concentrations over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how we detect, monitor, and treat many important diseases.

Appendicitis in late adulthood is characterized by a delay in treatment, high perforation rates, and unfavorable outcome parameters, all mutually correlating. Older patients with acute abdominal pain are high-risk patients, unlike their younger counterparts. They have to be clinically evaluated by experienced surgeons within a narrow time margin. The problem of late presentation and/or referral should be addressed, perhaps by education of primary care physicians and the public.