Lars Wieslander

A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels

Abstract Defined RNA fractions can be recovered from low gelling temperature agarose gels by a combination of agarose melting at 65°C and phenol extraction. By this approach RNA molecules up to a size of 37 kb can be eluted undegraded with a recovery of 60–90%. The method is applicable also to DNA and the eluted DNA can be correctly cleaved with restriction endonucleases as shown for λDNA using Eco RI.

The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm

The DEAD box RNA helicase Dbp5 is essential for nucleocytoplasmic transport of mRNA–protein (mRNP) complexes. Dbp5 is present mainly in the cytoplasm and is enriched at the cytoplasmic side of nuclear pore complexes (NPCs), suggesting that it acts in the late part of mRNP export. Here, we visualize the assembly and transport of a specific mRNP particle, the Balbiani ring mRNP in the dipteran Chironomus tentans, and show that a Dbp5 homologue in C.tentans, Ct‐Dbp5, binds to pre‐mRNP co‐transcriptionally and accompanies the mRNP to and through the nuclear pores and into the cytoplasm. We also demonstrate that Ct‐Dbp5 accumulates in the nucleus and partly disappears from the NPC when nuclear export of mRNA is inhibited. The fact that Ct‐Dbp5 is present along the exiting mRNP fibril extending from the nuclear pore into the cytoplasm supports the view that Ct‐Dbp5 is involved in restructuring the mRNP prior to translation. Finally, the addition of the export factor Dbp5 to the growing transcript highlights the importance of the co‐transcriptional loading process in determining the fate of mRNA.

In situ transcription and splicing in the Balbiani ring 3 gene

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co‐transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20–25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi‐intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well‐defined unit in situ and that the C‐terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.

The Balbiani Ring Multigene Family: Coding Repetitive Sequences and Evolution of a Tissue-Specific Cell Function

Publisher Summary This chapter discusses the current information about a specific gene family, the Balbiani ring (BR) multigene family. This gene family is found in the dipteran species Chironomus tentans, where its expression represents a major part of the tissue-specific function of the salivary glands. It is interesting for two reasons. First, it exemplifies how a tissue-specific function, involving the interaction of many different proteins, has evolved from a common gene ancestor, by sequence duplications and divergence of coding as well as of regulatory regions of the duplicated genes. Second, it is an example of how coding sequences may be reduplicated, within individual genes, to produce arrays of coding sequences, upon which recombination events operate, to continuously remodel the repetitive structure. The BR multigene family reflects the underlying mechanisms that form and then shape all the duplicated sequences in eukaryotic genomes. It, therefore, serves as a useful example, from which knowledge about evolution, by sequence duplication and remodeling of coding repetitive sequences, can be obtained.

The Balbiani ring 3 gene in Chironomus tentans has a diverged repetitive structure split by many introns.

A set of approximately 15 secretory proteins is synthesized by the salivary gland cells in the midge Chironomus tentans. These proteins are secreted but do not form insoluble fibers until they are transported out of the gland lumen. A Balbiani ring (BR) gene family consisting of four genes (BR1, BR2.1, BR2.2 and BR6) have previously been shown to encode four of these proteins, sp-I a to d, with relative molecular weights of 1 x 10(6). Each BR gene contains an uninterrupted block in which about 100 repeats are tandemly arranged. The repeats are virtually identical and efficient homogenization mechanisms must operate within each block. Here we describe a new BR gene, the BR3 gene, which according to structural similarities may belong to the BR gene family, but at the same time exhibits a strikingly different structure. The gene encodes a 10.9 kb transcript that contains 38 introns and is spliced into a 5.5 kb mRNA. The mRNA is translated into a cysteine-rich 185 kDa major component of the gland secretion. The coding sequence in the gene is built from diverged repeats in which mainly the cysteine codons are preserved and the sequence is split by the introns into 17 to 678-bp long exons. The introns are located at defined positions in relation to the repeat structure. In sharp contrast to the uninterrupted array of identical repeats in the BR1-BR6 genes, the repeats in the BR3 gene are not efficiently homogenized and have diverged extensively from each other. We propose that the splitting of the repeat structure into variable sized exons prevents homogenizations dependent on unequal aligning of homologous sequences.

A hierarchic arrangement of the repetitive sequences in the Balbiani ring 2 gene of Chironomus tentans

One cloned cDNA sequence, pCt63, was used to characterize the repeated structure of the Balbiani ring 2 gene in Chironomus tentans. Although small in size (0.63 kb), the cDNA insert corresponds to a large portion (25 kb) of the BR2 gene (37 kb). Southern blotting experiments suggested that a large part of the BR2 gene consists of tandemly repeated units, each about 215 bp. Sequence analysis of the cDNA confirmed the repeated nature of the BR2 gene and revealed the internal structure of the repeat unit. Each such unit is composed of two regions of approximately equal length; one is highly ordered and built from about six 18 bp repeats, each consisting of a slightly diverged 9 bp duplication. The recorded hierarchic arrangement of the repetitive sequences in the BR2 gene and a specific pattern of base substitutions along the gene have enabled us to propose how a major part of the giant BR2 gene has evolved from a short primordial sequence, 110-120 bp in length.

Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions

Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3′ end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.

Conserved and nonconserved structures in the secretory proteins encoded in the Balbiani ring genes ofChironomus tentans

SummaryThe large, repetitive Balbiani ring (BR) genes, BR 1, 2, and 6, inChironomus tentans originated from a short ancestral sequence and have all evolved according to analogous amplification schemes. We analyzed the structures of the BR-encoded secretory proteins and defined the parts that have been conserved during the evolutionary process. The BR products show striking similarities, with the BR 1 and BR 2 products being more similar to each other than to the BR 6 product. In the constant (C) region of the repeat units, 7 of the 30 amino acid residues are strictly conserved; 4 of these are the cysteine residues. The subrepeat (SR) regions of all the BR products are dominated by repeated tripeptide elements rich in proline and charged amino acid residues. Most of the amino acid replacements in both regions are conservative. Secondary structure predictions suggested that the C regions of the BR 1 and BR2 products have several elements of secondary structure: an α-helix, a β-strand, and one or two reverse turns, as in “globular structures.” The prediction for the C region of the BR 6 product is similar but lacks a β-strand. The predictions for the intervening SR regions appear less conclusive, but are clearly different from those for the C regions, and suggest regular structures not differing in their conformational elements. The SR regions evolved from an ancestor sequence similar to the C region; thus, the BR products seem to represent an example of evolution from one structure to two differently folded products. It is proposed that the alignment and polymerization of the long BR proteins could be promoted by the repetitive structure of the molecules, due to the possibility of forming disulfide bridges between half-cystine residues and electrostatic interactions between the charged residues of the SR regions. The divergence among the BR products is discussed in relation to possible functional differences among the members of the BR gene family.

Secretory Proteins of Chironomus Salivary Glands: Structural Motifs and Assembly Characteristics of a Novel Biopolymer.

Salivary glands of Chironomus synthesize a family of at least ten secretory proteins that can be grouped into three size classes: the large (about 1000 kDa), intermediate (100- to 200 kDa), and small (less than 100 kDa). After synthesis, secretory proteins undergo a dramatic transformation to form a novel biopolymer. Secretory proteins accumulate in the central lumen of the gland, forming dissociable complexes that appear as a network of smooth fibrils and multistranded beaded fibers. When secretory protein complexes are extruded through the secretory duct, the fibers become oriented in parallel arrays; when these parallel arrays of fibers emerge from the mouth of larvae they are an insoluble, silk-like thread. Regulation of secretory protein-coding gene expression determines which secretory proteins are synthesized, thus, the composition of silk threads. At least two types of threads are produced: larval silk is used to construct tubes for protective housing and assist with feeding; prepupal silk is used to construct tubes for larval/pupal ecdysis (pupation). Variations in composition presumably contribute to different mechanical properties of larval and prepupal silk threads. Since the macroscopic physical properties of polymerized silk most likely reflect the microscopic structure and interaction of secretory proteins, it becomes important to learn the principles which govern secretory protein assembly at the molecular level. Which secretory proteins interact and what are the sites used for intraportein and protein-protein interactions during the assembly of this biopolymer? All eight secretory proteins characterized thus far contain tandemly repeated peptide sequences (ranging from 14-90 amino acids in length) and/or a periodic distribution of Cys residues. These motifs appear to be unique; no other biopolymer has either the repeated peptide sequences or composite structure of chironomid silk threads. The evolutionary conservation of motifs within repeats and among different secretory proteins suggests that the sequences and three-dimensional structures of the motifs may be important for assembly of secretory proteins into complexes, oriented fibers, and silk threads. Further study of secretory protein assembly will bring us closer to understanding how this silk assembles in vivo. By learning principles that nature employs to construct such a novel composite biopolymer, it may become feasible to design and produce new classes of fibers or biomolecular materials with distinctive properties that are currently unavailable.

Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly.

Summary In vivo UV crosslinking identified numerous preribosomal RNA (pre-rRNA) binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0–A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30, and snR10, which are required for cleavage at A0–A2. The NTD was crosslinked to sequences flanking A3 and to the RNA component of ribonuclease MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural proteins and nucleoside triphosphatases. A key role in coordinating preribosomal assembly and processing was confirmed by chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and preribosome compaction greatly reduced.