hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells

Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H' was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H', and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.     

Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds

Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (K_a > 10¹¹ M^− 1) and, more weakly, bovine α-chymotrypsin (K_a ≈ 10⁴–10⁶ M^− 1). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.     

A biomechanical role for perlecan in the pericellular matrix of articular cartilage

Chondrocytes are surrounded by a narrow pericellular matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.     

HuR and Translation-The Missing Linc(RNA)

In this issue, Yoon et?al. (2012) reveal that HuR influences decay of a long noncoding RNA (lincRNA-p21) that in turn hybridizes to mRNA targets and represses their translation-an indirect, but elegant explanation for the impact of HuR on translation efficiency.     

Chemical synthesis of new trypsin, chymotrypsin and elastase inhibitors by amino-acid substitutions in a trypsin inhibitor from squash seeds (CMTI III)

Five new analogues of squash trypsin inhibitor CMTI III were obtained by the solid-phase method, exhibiting antitrypsin, antichymotrypsin or antielastase activity. Modification in the reactive site region changes dramatically the specificity, whereas substitution in non-contact positions facilitates the refolding of the reduced form of the peptides and does not have a significant influence on the association equilibrium constants of the inhibitor analogues with cognate enzymes.     

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.     

Cleavage site determinants in the mammalian polyadenylation signal.

Using a series of position and nucleotide variants of the SV40 late polyadenylation signal we have demonstrated that three sequence elements determine the precise site of 3-end cleavage in mammalian pre-mRNAs: an upstream AAUAAA element, a down-stream U-rich element consisting of five nucleotides, at least four of which are uridine, and a nucleotide preference at the site of cleavage in the order A > U > C >> G. Cleavage occurs no closer than 11 bases, but no further than 23 bases from the AAUAAA element. The downstream U-rich element is usually located 10-30 bases from the cleavage site. The relative position of the AAUAAA and the U-rich elements define the approximate region within a 13 base domain in which cleavage will occur. The exact position of cleavage is then determined by the local nucleotide sequence in the order of preference noted above. This model accounts for nearly three quarters of polyadenylation signals surveyed and is consistent with previous experimental observations.     

Synthesis of an elastase inhibitor by monosubstitution of arginine-5 with valine at the reactive site in a trypsin inhibitor from squash seeds (CMTI III)

Using the solid-phase procedure an analog of trypsin inhibitor CMTI III containing Val5 instead of Arg5 in position P1, was synthesized. The substitution in only this one position P1 increased the affinity of synthetic inhibitor to porcine pancreatic elastase and human leukocyte elastase by the factor of 10(3) and 10(7), respectively.