Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

Acute suppurative renal inflammation.

Diagnostic and therapeutic aspects of acute suppurative renal inflammation are discussed in connection with 374 cases. Despite negative X-ray and laboratory findings, patients were submitted to operative exploration and 27% of them revealed subcapsular abscesses. Anamnestic facts (fever, shiver, etc.) and closely observed progression in the patient's morbid condition are factors to indicate surgery. Better results from the second part of the observation period (1981-1989) are due partly to better timing of the surgical intervention, partly to modern, highly effective chemotherapy. The mortality rate of acute suppurative renal inflammation dropped from 11 to 3.5%.

Immunological characterization and intracellular localization of trans-spliceosomal small nuclear ribonucleoproteins in Trypanosoma brucei.

Polyclonal antibodies were raised against purified protein components of the U2 small nuclear ribonucleoprotein (snRNP) from Trypanosoma brucei. Through immunoblot and immunoprecipitation analyses three antisera were characterized that reacted specifically with U2 snRNP proteins of molecular weights 40,000 (anti-40K) and 16,500 (anti-16.5K), and with each of four proteins of molecular weights 14,000, 12,500, 10,000, and 8,500 (anti-CP). Anti-40K antibodies specifically immunoprecipitated the U2 snRNP from trypanosomal extracts, whereas anti-CP antibodies recognized several snRNPs, including the SL RNP and the U2 and U4/U6 snRNPs; in addition, minor RNAs were detected, suggesting that a family of snRNPs with common or related protein components exists in trypanosomes. None of these antibodies cross-reacted significantly with total mammalian snRNP proteins, indicating that the trypanosomal snRNP proteins are immunologically distinct from their mammalian counterparts. Using immunofluorescence microscopy, the snRNP proteins exhibited a differential cellular distribution. Whereas the 40-kDa protein is localized exclusively in the nucleus, with the nucleolus being excluded, a fraction of the common proteins also resides in the cytoplasm.

Heavy metal ion-induced changes of ribosomal RNA synthesis in Synechococcus sp. strain PCC 6301, a cyanobacterium.

We examined the effect of cadmium, zinc, copper and mercury ions on the rate of total RNA synthesis and on the accumulation of ribosomal RNAs in Synechococcus sp. PCC 6301, an obligate photoautotrophic cyanobacterium. It was found that treatment of cells with growth-inhibitory concentrations of cadmium and zinc ions severely decreased the rate of total RNA synthesis both under light (growing) and dark (non-growing) conditions, while copper and mercury ions had different inhibitory effects under these conditions. Moreover, cadmium and zinc ions substantially inhibited the "normal" processing of high-molecular-mass rRNAs and the in vivo postmaturational cleavage of 23S rRNA in the light and interfered with the accumulation of dark-specific RNAs of 0.33 x 10(6), 0.24 x 10(6) and 0.16 x 10(6) daltons in the dark. On the other hand, copper and mercury ions had a similar effect in the light, but did not abolish the accumulation of dark-specific, rRNA-derived, RNAs in the dark.

Alterations in the accumulation of adenylylated nucleotides in heavy-metal-ion-stressed and heat-stressed Synechococcus sp. strain PCC 6301, a cyanobacterium, in light and dark.

Heavy-metal-ion- (Cd2+, Cu2+, Pb2+, Hg2+ and Zn2+) or heat (50 degrees C)-stress treatments of the unicellular cyanobacterium Synechococcus sp., strain PCC 6301, under both light and dark conditions led to the accumulation of bis(5'-nucleosidyl)oligophosphates: Ap4A, Ap4G, Ap3A, Ap3G and Ap3Gp2. Under light regimens, the accumulation of Ap4A and Ap4G is more characteristic of heavy-metal-ion-stressed cells, whereas the accumulation of Ap3A, Ap3G and Ap3Gp2 is the dominant feature of heavy-metal-ion or heat-shock treatment during energy deprivation (i.e. in the dark). This accumulation of bisnucleoside oligophosphates supports a model whereby the adenylylated nucleotides are synthesized by the backward reaction of tRNA-aminoacyl synthetases. These nucleotides may also act to switch or modulate cyanobacterial responses under various environmental stress conditions.

Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301.

The response to heat shock at 47 degrees C was examined in the cyanobacterium (blue-green alga) Synechococcus sp. strain PCC 6301. On heat shock, the growth of the cells decreased and they preferentially synthesized a limited number of polypeptides. The rate of synthesis of these proteins increased markedly in the early period of temperature shift up and gradually decreased afterwards. Among the proteins greatly affected by temperature shift up were those with apparent molecular weights of 91,000 (91K), 79K, 78K, 74K, 65K, 64K, 61K, 49K, 45K, 24K, 22K, 18K, 16K, 14K, 12K, and 11.4K, based on their mobilities in sodium dodecyl sulfate-polyacrylamide gels. From these initial studies on Synechococcus sp. strain PCC 6301 we conclude that in cyanobacteria a heat shock response similar to that known to occur in other eucaryotes and procaryotes might exist.

Affinity purification of Trypanosoma brucei small nuclear ribonucleoproteins reveals common and specific protein components.

We have developed a procedure for the affinity purification of small nuclear ribonucleoproteins (snRNPs) of Trypanosoma brucei (U2 and U4/U6 snRNPs), which are essential for trans splicing. Each of these snRNPs can be specifically and efficiently selected from T. brucei extracts through biotinylated antisense 2'-O-methylated RNA oligonucleotides immobilized on streptavidin-agarose. Protein analysis revealed a set of five low molecular weight polypeptides common to the U2 and U4/U6 snRNPs and the spliced leader RNP. In addition, several U2 and U4/U6 snRNP-specific protein components were identified. Using monoclonal antibodies against human snRNP proteins, we could not detect any significant cross-reaction with the trypanosomal U2 snRNP proteins. Thus, the trypanosomal snRNPs exhibit principal differences from the higher eukaryotic snRNPs not only in their RNA but also in their protein components.

Spliced leader-associated RNA of trypanosomes. Sequence conservation and association with protein components common to trans-spliceosomal ribonucleoproteins.

A spliced leader-associated (SLA) RNA recently identified in Trypanosoma brucei has been proposed as a U5 RNA analog in trans-splicing, based on in vivo psoralen cross-linking to the spliced leader (SL) RNA and a short stretch of homology to a conserved U5 RNA sequence. Here we show that the SLA RNA is in the form of an RNP containing common proteins that also occur in the known trans-spliceosomal snRNPs of T. brucei, the SL, U2, and U4/U6 snRNPs. Therefore the SLA RNP is a member of a larger family of related RNPs. The immunoprecipitation of the SLA RNA by antibodies against common proteins does not require an association between the SLA RNA and the SL RNP. To identify sequences of potential functional importance and to establish a secondary structure model, we have sequenced the SLA RNA and cloned its gene from a related trypanosomatid, Leptomonas seymouri. A sequence comparison between the T. brucei and L. seymouri SLA RNAs reveals that the U5-homologous sequence element is not conserved.

Carcinoma of the penis as a metastasis.

A case of carcinoma of the penis is reported where the tumour was metastatic in origin. The patient recovered postoperatively. Based on the histological study of the removed tumour, suspicion of bronchial cancer arose to be the primary tumour on the right side. Subsequently, despite the physician's advice, the patient left the department of pulmonology for home and so no further pulmonological examination and treatment were made. One year postoperatively, local recurrence in the penis could not be noted.

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.

Assembly of the U2 small nuclear ribonucleoprotein from Trypanosoma brucei. A mutational analysis.

trans-Splicing in trypanosomes requires the functions of U2 and U4/U6 small nuclear (sn) RNPs. We have analyzed protein binding and assembly of the Trypanosoma brucei U2 snRNP, using specific antibodies against U2 snRNP proteins and in vitro reconstitution assays of U2 deletion derivatives and human-trypanosome hybrid RNAs. Stable binding of both the U2-specific 40-kDa and the common proteins requires only the 3'-terminal domain (stem-loop IIb, single-stranded region, and stem-loop IV), with loop IV providing the critical sequence determinant; stem-loop IV suffices for binding of the 40 kDa-protein, but not of the common proteins; surprisingly, the sequence of the "Sm-analogous" single-stranded region between stem-loops IIb and IV is not essential for protein binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the assembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP assembly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from that of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3'(2')-diphosphate 5'-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.

Plant small nuclear RNAs. V. U4 RNA is present in broad bean plants in the form of sequence variants and is base-paired with U6 RNA.

U4 RNA, which is known to play an indispensable role in pre-mRNA splicing, is present in plant nuclei, has a canonical m3 2,2,7 G cap at its 5' end and is associated with U6 RNA in snRNP particles. It occurs in broad bean in the form of a number of sequence variants. Two of these were sequenced: U4A RNA is 154 and U4B RNA is 152 nucleotides long. Sequence similarity of broad bean U4B RNA is 94 per cent to broad bean U4A RNA, 65 per cent to rat U4A RNA, 61 per cent to Drosophila U4A RNA and 50 per cent to snR14, the U4 RNA equivalent of the yeast Saccharomyces cerevisiae. Sequence conservation is much more pronounced in the 5' half of the molecule than in its 3' half. The secondary structure of both variants of broad bean U4 RNA perfectly fits with that of all other U4 RNAs sequenced so far. Nucleotide changes between broad bean U4A and U4B RNAs are restricted to molecular regions that affect the thermodynamic stability of these molecules. A model is proposed for the base pairing interaction of broad bean U4 RNA with broad bean U6 RNA. This is the first report on the structure of a plant U4 RNA.

The trans-spliceosomal U2 snRNP protein 40K of Trypanosoma brucei: cloning and analysis of functional domains reveals homology to a mammalian snRNP protein.

Through immunoscreening we have isolated a cDNA encoding the trans-spliceosomal U2 snRNP-specific 40 kDa protein of Trypanosoma brucei. The protein has a predicted molecular weight of 36.6 kDa and shows 31% amino acid identity with the human U2 snRNP A' protein of 28.4 kDa. The homology between the trypanosome and human protein sequences is restricted to the N-terminal half where they share a series of six leucine repeat motifs. Sequence alignment revealed three 40K-specific regions: a C-terminal extension and two insertions, one of which makes up a seventh leucine repeat. Bacterially expressed 40K protein efficiently bound RNA by itself in a nonspecific manner; this general RNA binding activity was located to a region in the C-terminal half overlapping with the leucine repeat domain. U2 RNA-specific interaction required the presence of other trypanosome proteins and depended upon the loop IV sequence of U2 RNA. Deletion analysis of the 40K protein demonstrated the leucine repeats, including the 40K-specific, seventh repeat, to be essential for specific U2 RNP assembly, most likely through their role as an interface for protein-protein interaction.

The spliceosomal snRNP core complex of Trypanosoma brucei: cloning and functional analysis reveals seven Sm protein constituents.

Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.