The small nuclear 7SK RNA negatively controls transcription by inactivating positive transcription elongation factor b (P-TEFb) and is an integral component of Tat-dependent and independent HIV-1 transcription initiation complexes. 7SK RNA has recently been shown to also directly control HMGA1 transcription activity. HMGA1 is a master regulator of gene expression and its deregulation is associated with virtually any type of human cancer. The degree of HMGA1 over-expression thereby correlates with tumor malignancy and metastatic potential. 7SK snRNA directly interacts through its loop2 (7SK L2) with the first A/T-hook DNA binding motif of HMGA1. We have developed several 7SK L2 RNA chimera with the Epstein Barr Virus expressed RNA 2 (EBER2) to target HMGA1 function in transcription regulation. The efficiency of interfering with HMGA1 transcription activity by the chimeric 7SK L2-EBER2 fusions by large exceeds the efficiency of 7SK wild-type RNA due to the stronger EBER2 promoter activity. Furthermore, the 7SK L2-EBER2 chimera do not interfere with P-TEFb controlled transcription elongation or the formation of 7SK sn/hnRNPs. The comparison of the effects of wild-type 7SK RNA on cellular transcriptome dynamics with those induced by the two 7SK L2 mutants as well as the changes in gene expression following inhibition of HMGA1 allow the identification and characterization of HMGA1-dependent and independent effects of 7SK snRNA. We furthermore also present evidence for P-TEFb and HMGA1-independent 7SK RNA L2 regulatory activity.