RNA polymerase II transcripts, heterogeneous nuclear RNAs (hnRNAs), associate in the nucleus with specific proteins that bind premessenger RNA (hnRNP proteins) and with small nuclear ribonucleoprotein particles (snRNPs). These hnRNA-hnRNP-snRNP complexes assemble on nascent transcripts and hnRNA is processed to mRNA in them. HnRNP proteins have been localized to the nucleoplasm and their functions were presumed to be limited to nuclear events in mRNA biogenesis. It was proposed that an exchange of hnRNP for mRNA-binding proteins accompanies transport of mRNA from the nucleus to the cytoplasm. We show here that several of the abundant hnRNP proteins, including A1, shuttle between the nucleus and the cytoplasm. HnRNP proteins may thus also have cytoplasmic functions. Furthermore, when in the cytoplasm, A1 is bound to mRNA and RNA polymerase II transcription is necessary before it can return to the nucleus. We propose that the cytoplasmic ribonucleoprotein complex of mRNA with hnRNP proteins is the substrate of nuclear-cytoplasmic transport of mRNA.

Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm

We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

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Dsif, a novel transcription elongation factor that regulates rna polymerase ii processivity, is composed of human spt4 and spt5 homologs

Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.

Structure and functions of nucleolin

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

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Transcription elongation factor P-TEFb is required for HIV-1 Tat transactivation in vitro

The elongation phase of transcription by RNA polymerase II is one of the many steps during the generation of mature mRNAs that is subject to regulation. Shortly after initiation, RNA polymerase II comes under the control of negative transcription elongation factors, generally termed N-TEFs, and enters abortive elongation (51). During this postinitiation process, only short transcripts are generated that may be prematurely terminated. These short transcripts arise from transcription of many genes, including c-myb, c-myc, c-fos, HSP70, and the human immunodeficiency virus (HIV) long terminal repeat (LTR), and are normally subject to rapid degradation (3, 63). Escape from the action of N-TEF requires the action of at least one positive transcription elongation factor (P-TEF), eventually identified as P-TEFb (52). P-TEFb allows the transition into productive elongation, producing long transcripts from which mRNAs are derived. In this way, the fraction of initiating RNA polymerase II molecules that produce full-length transcripts is controlled by a selection process that occurs early in the elongation phase of the transcription cycle. After the transition is made into productive elongation, the efficiency of elongation may be influenced by additional factors, including S-II, TFIIF, ELL, and elongin (62, 65).

P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II.

Electrophoretic characterization of nucleolar RNA from Chironomus tentans salivary gland cells.

A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis.

Nuclear casein kinase II (CK II) was purified from an epithelial cell line ofChironomus tentans and characterized. The intracellular distribution of CK II and its two intracellular subunits (α and β) was analysed by immunoblotting. The apparent molecular weights of the α and β subunits were estimated to be 36 and 28 kDa, respectively. Like other purified CK II preparations, CK II fromChironomus tentans is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and by the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual α and β subunit pools could be detected. More than 85% of the total immunostainable α subunit and essentially all immunoreactive individual β subunit and heterooligomeric enzyme molecules were localised to the nucleus. Unexpectedly, more than 80% of this nuclear α subunit was insoluble in 0.35 M NaCl, while all individual β subunit and heterooligomeric enzyme molecules were solubilized under the same conditions. Of the 0.35 M NaCl soluble kinase fractions, the active multisubunit form of CK II precipitated in 50% (NH4)2SO4 and could thus be separated from the free β subunit, which precipitated at 60% and 80% (NH4)2SO4. These results suggest that a major portion of the nuclear CK II α subunit does not form heterooligomeric structures with the β subunit, but binds tightly to nuclear components.

A majority of casein kinase II alpha subunit is tightly bound to intranuclear components but not to the beta subunit.

The presence of heterogeneous RNA of high molecular weight has been demonstrated on the giant chromosomes, in the nuclear sap and in the cytoplasm of the salivary glands in Chironomus tentans. The kinetic properties of this heterogeneous RNA have also been outlined in some detail. — Salivary glands were incubated for different time intervals (20, 45 and 180 min) in haemolymph, supplemented with tritiated cytidine and uridine. The different cellular components were isolated by micromanipulation and RNA extracted with an SDS-pronase solution and analysed with electrophoresis in agarose. — Heterogeneous, high molecular weight RNA with a peak around 35 S was saturated with label on chromosome I, II and III in 45 min, although the synthetic capacity was unchanged during at least 180 min incubation. This indicated a complete turnover of heterogeneous RNA on the chromosomes in less than 45 min. The turnover time in the giant puffs (the so called Balbiani rings) on the fourth chromosome, was even shorter and estimated to less than 30 min. No shift in the electrophoretic pattern of this heterogeneous RNA was found to occur on the chromosomes during long incubation times or during actinomycin D experiments. These labelling characteristics of heterogeneous RNA on the chromosomes indicate that all the different molecules in the heterogeneous RNA have a similar and rapid turnover. A conversion to smaller, stable molecules was excluded. — Heterogeneous RNA of a distribution corresponding to that on the chromosomes was found in the nuclear sap and also in the cytoplasm. The activity in both these cellular compartments increased between 45 and 180 min incubation. The distribution pattern for high molecular weight RNA was in all experiments similar on the chromosomes, in the nuclear sap and in the cytoplasm. It appears that at least a considerable part of the high molecular weight RNA leaves the chromosomes to enter the nuclear sap and lateron to some extent the cytoplasm in this high molecular form. Stable molecules of smaller size (6–15 S) did not appear during 180 min incubation. The data indicate, however, also a substantial breakdown of heterogeneous RNA to acid soluble products during this time.

Chromosomal RNA synthesis in polytene chromosomes of Chironomus tentans.

Rapidly labelled RNA in Balbiani ring 2 on chromosome IV in the salivary glands of Chironomus tentans was investigated. This RNA is likely to be transcribed from only one chromosomal band, supposed to be a single operational unit in these polytenic cells (Beermann, 1966).

Endre Egyházi

Papers

Electrophoretic characterization of nucleolar RNA from Chironomus tentans salivary gland cells.

A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis.

A majority of casein kinase II alpha subunit is tightly bound to intranuclear components but not to the beta subunit.

Chromosomal RNA synthesis in polytene chromosomes of Chironomus tentans.

RNA synthesis in a Balbiani ring in Chironomus tentans salivary gland cells.