hnRNP C is required for postimplantation mouse development but Is dispensable for cell viability.

The hnRNP C1 and C2 proteins are among the most abundant proteins in the nucleus, and as ubiquitous components of RNP complexes, they have been implicated in many aspects of mRNA biogenesis. In this report, we have characterized a null mutation induced in embryonic stem cells by insertion of the U3His gene trap retrovirus into the first intron of the hnRNP C1/C2 gene. cDNAs encoding murine hnRNP C1 and C2 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. A human consensus sequence, generated from over 400 expressed sequence tags, suggests two revisions to the previously published human sequence. In addition, alternatively spliced transcripts, expressed only by the murine gene, encode four novel proteins: variants of C1 and C2 with either seven additional amino acids or one fewer amino acid in a region between the oligomerization and C-terminal acidic domains. The disrupted gene was transmitted into the germ line and is tightly linked to a recessive, embryonic lethal phenotype. Homozygous mutant embryos fail to develop beyond the egg cylinder stage and are resorbed by 10.5 days of gestation, a phenotype consistent with a fundamental role in cellular metabolism. However, hnRNP C1 and C2 are not required for cell viability. Embryonic stem cell lines established from homozygous mutant blastocysts did not express detectable levels of either protein yet were able to grow and differentiate in vitro, albeit more slowly than wild-type cells. These results indicate that the C1 and C2 hnRNPs are not required for any essential step in mRNA biogenesis; however, the proteins may influence the rate and/or fidelity of one or more steps.

The antiterminator RNA of phage HK022.

The put sites of phage HK022 increase the processivity of transcription and thereby promote the expression of viral genes that are located downstream of transcription terminators. RNA polymerase molecules that have traversed a put site are converted to a terminator-resistant form by the put transcript. We analyzed the structure and function of put transcripts by determining the effects of put mutations on terminator read-through, and by probing wild-type and mutant put RNAs with structure-specific nucleases. The results support the prediction that the secondary structure of the active transcript consists of two hairpin stems that are separated by a single unpaired base. The identity of bases in certain bulges and internal loops is important for activity, while that of most bases in the terminal loops is not. Many bases in the stems can be replaced with little or no effect on activity provided that base-pairing is maintained.

Transcripts that increase the processivity and elongation rate of RNA polymerase.

Transcripts encoded by the cis-acting antitermination sites (put sites) of lambdoid phage HK022 promote readthrough of downstream transcription terminators. Proper conformation of the transcripts is essential for activity, since put mutations that prevent the formation of predicted RNA stems prevented antitermination, and suppressor mutations that restore the stems restored antitermination. Antitermination does not appear to require proteins other than RNA polymerase, since put-dependent readthrough of multiple sequential terminators was observed in a purified transcription system consisting of template, polymerase, substrates, and buffer. Transcription of put also increased the elongation rate of polymerase, very likely by suppressing pausing. A mutation that alters the zinc-finger region of the beta' subunit of polymerase specifically prevented the put-dependent increases in terminator readthrough and elongation rate. The simplicity of HK022 antitermination contrasts with that of other known antitermination pathways. We propose that the central effector is a transcript that directly alters the elongation properties of RNA polymerase.