No abstract

Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.

No abstract

Non-coding (nc) RNAs are increasingly recognized to play important regulatory roles in eukaryotic gene expression. The highly abundant and essential 7SK ncRNA has been shown to negatively regulate RNA Polymerase II transcription by inactivating the positive transcription elongation factor b (P-TEFb) in cellular and Tat-dependent HIV transcription. Here, we identify a more general, P-TEFb-independent role of 7SK RNA in directly affecting the function of the architectural transcription factor and chromatin regulator HMGA1. An important regulatory role of 7SK RNA in HMGA1-dependent cell differentiation and proliferation regulation is uncovered with the identification of over 1500 7SK-responsive HMGA1 target genes. Elevated HMGA1 expression is observed in nearly every type of cancer making the use of a 7SK substructure in the inhibition of HMGA1 activity, as pioneered here, potentially useful in therapy. The 7SK-HMGA1 interaction not only adds an essential facet to the comprehension of transcriptional plasticity at the coupling of initiation and elongation, but also might provide a molecular link between HIV reprogramming of cellular gene expression-associated oncogenesis.